Polypeptide analogs of apolipoprotein E, diagnostic systems and methods using the analogs

ABSTRACT

The present invention contemplates a multimeric polypeptide capable of mimicking the ability of apo E to induce differentiated cellular function. The repeating unit of the polypeptide has an amino acid residue sequence corresponding to that represented by the formula LRXLRKRLLX. Also contemplated is a method for treating hypercholesterolemia in a patient, which method comprises administering to the patient an LDL plasma concentration-reducing amount of the polypeptide. Described as well, is the use of the polypeptide in preparing diagnostic antibodies, and their use in diagnostic systems and methods for detecting apo E antigens in vascular body fluids.

This invention was made with government support under NationalInstitutes of Health Contract HL-35297 R01. The government has certainrights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.07/769,629, filed Sep. 30, 1991, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 07/625,093, nowabandoned, filed Dec. 10, 1990, which is a continuation-in-part of U.S.patent application Ser. No. 07/540,363, now U.S. Pat. No. 5,168,045,filed Jun. 18, 1990 which is a continuation-in-part of U.S. patentapplication Ser. No. 07/485,158, filed Feb. 26, 1990, now U.S. Pat. No.5,182,364, which is a continuation-in-part of U.S. patent applicationSer. No. 07/395,732, filed Aug. 18, 1989, now U.S. Pat. No. 5,177,189,the disclosures of which are hereby incorporated by reference.

TECHNICAL FIELD DESCRIPTION

The present invention relates to polypeptides capable of mimicking theability of apolipoprotein (apo) E to induce differentiated cellularfunction. More particularly, the present invention relates to analogs ofApo E useful for inhibiting lymphocyte proliferation and/or ovarianandrogen secretion. The present invention also relates to enhancement ofcholesterol clearance mediated by these receptor-binding(receptor-competent) polypeptides. In that regard, the present inventionrelates to the use of synthetic tandem peptides to modulate hepatic celluptake of cholesterol-containing proteins and to diagnostic methodsusing these peptides and their antibodies to evaluate efficacy of thesetherapeutic measures.

BACKGROUND

Lipoproteins are the primary carriers of plasma cholesterol. They aremicellar lipid-protein complexes (particles), having a surface filmcomprised of one or more proteins associated with polar lipids, thatsurrounds a cholesterol-containing core. Original classification oflipoproteins was based on their buoyant densities as measured byultracentrifugation. Accordingly, four major density classes have beenrecognized, and subclasses within these exist.

The first class comprises the chylomicrons. They are the largest of thelipoproteins and are rich in triglycerides. The site of origin of thechylomicrons is the intestine. When chylomicrons are exposed to plasmaor high density lipoprotein (HDL) in vitro, much of their complement ofA apolipoproteins is lost, and C and E apolipoproteins are acquired.Chylomicrons also contain apolipoprotein B-48.

The second class of lipoproteins, the very low density lipoproteins(VLDL), is comprised of particles made in the liver and involved intriglyceride metabolism and transport from the liver. Theapolipoproteins, apo B-100 and apo E, are the major constituents of theVLDL particle.

The third lipoprotein class, comprising low density lipoproteins (LDL),is a specific product of the catabolism of VLDL. The predominantapolipoprotein in LDL particles is apolipoprotein B-100, or apo B-100.

The fourth class, high density lipoprotein (HDL) contains two majorapolipoproteins, apo A-I and apo A-II. One function of apo A-I is theactivation of the plasma enzyme, lecithin-cholesterol acyltransferase,which is required for the esterification of free cholesterol on HDL fortransport to the liver.

Plasma cholesterol is regulated in part by the LDL receptor and in parton the ability of the lipoprotein to carry cholesterol and bind the LDLreceptor. Hofmann, et al., Science, 239:1277 (1988). This receptor isfound on the surface of all cells where it mediates binding andinternalization of the cholesterol-rich lipoproteins that providemembrane cholesterol; Brown, et al., J. Clin. Invest., 55:783 (1975);Goldstein, et al., Methods Enzymol., 98:241 (1983); and in somespecialized cells substrate cholesterol for the production of bile acidsor steroid hormones; Gwynne, et al., Endocr. Rev., 3:299 (1982). LDLreceptor expression on cells is inversely regulated by the circulatingconcentration of LDL, i.e., the higher the circulating LDL the fewer LDLreceptors on the cell surface Hofmann, et al., Science, 239:1277 (1988).LDL binding to the LDL receptor has been the focus of much researchbecause of its importance in regulating the level of plasma cholesterol,which is considered a major risk factor for the development of coronaryartery disease; Brown, et al., Scient. Amer., 251:58 (1984).

LDL binding to the LDL receptor is believed to be dependent upon thespecies of apolipoprotein present in the lipoprotein particle.

When compared to apolipoproteins A-1 or B, the relative concentration ofapo E in plasma is low. However, apo E is instrumental in lipoproteinmetabolism in several ways. Mahley, et al., J. Lipid Res., 25:1277-1294(1984). It is a recognition site for several cellular lipoproteinreceptors, including hepatocyte receptors for chylomicron and VLDLremnants [Hui, et al., J. Biol. Chem., 259:860-869 (1984); Shelburne, etal., J. Clin. Invest., 65:652-658 (1980)], receptors for LDL on hepaticand extrahepatic cells [Hui, et al., J. Biol. Chem., 256:5646-5655(1981)] and receptors for VLDL on macrophages [Wang-Iverson et al.,Biochem. Biophys. Res. Commun., 126:578-586 (1985)].

Lipoproteins are cleared from the plasma by binding to high-affinityreceptors on liver cells and extrahepatic tissues such as the adrenalglands and ovaries. Kowal, R.C. et al., Proc. Natl. Acad. Sci. USA,86:5810-5814, (1989). The LDL receptor specifically binds apo B and apoE-bearing lipoproteins. R. W. Mahley, Science, 240:622 (1988). Thus, apoE is of clinical importance for its role in binding LDL receptor andfacilitating cholesterol clearance.

The LDL receptor-binding region of apo E has been mapped to an internalsequence including amino acid residues 140 to 160. Weisgraber, et al.,J. Biol. Chem. 258:12348 (1983). Additionally, apo E binds the LDLreceptor only when it is associated with a lipoprotein or phospholipid[Innerarity, et al., J. Biol. Chem., 254:4186 (1979)] and 4 apo Emolecules bind the LDL receptor with an affinity that is 10 to 25-foldgreater than the binding of a single molecule of apo B. R. W. Mahley,Science, 240:622 (1988).

Two distinct sets of receptors bind apo E-containing lipoproteins. TheLDL receptor [Yamamoto et al., Cell, 39:27-38 (1984)], 70% of which isthought to be located on hepatic cells, binds VLDL and apo E-containingremnants of chylomicrons. The existence of a second set of LDLreceptors, termed "remnant receptors", is inferred from studies showingthat the plasma clearance of apo E-containing chylomicron remnantsoccurs at normal rates in animals with genetically defective LDLreceptors.

Recently, an LDL receptor-related protein (LRP) has been found on thesurface of hepatic cells. Herz et al., EMBO, 7:4119-4127 (1988). LRPshares cysteine-repeat sequences with LDL and has been shown to bind andmediate the extracellular clearance of apo E-containing lipoproteins.Kowal, R.C. et al. Proc. Natl. Acad. Sci. USA, 86:5810-5814, (1989).

Apo E-enriched lipoproteins have also been described to have a functionin the immune system by inhibiting mitogen-or antigen-stimulatedlymphocyte proliferation in vitro and in vivo. In the ovary, apo Einhibits androgen production by LH-stimulated cultured theca andinterstitial cells; Dyer, et al., J. Biol. Chem., 263:10965 (1988).

In 1976 it was reported that a discrete lipoprotein fraction isolatedfrom normal human plasma inhibited mitogen- and allogeniccell-stimulated human lymphocyte proliferation in vitro (Curtiss et al.,J. Immunol., 116:1452, (1976)). This inhibitory plasma lipoprotein wastermed LDL-In for Low Density Lipoprotein-Inhibitor because the activefraction is localized to a less dense subfraction of total LDL ofdensity 1.006-1.063 g/ml. The characteristics of LDL-In-mediatedinhibition in vitro are as follows: LDL-In has comparable inhibitoryactivity for phytohemagglutinin (PHA), pokeweed mitogen (PWM), andallogenic cell-stimulated human lymphocyte proliferation. The inhibitoryactivity of LDL-In is non-toxic and independent of mitogenconcentration. Suppression by LDL-In is time dependent and approximately18 hr of exposure of the lipoprotein to the lymphocytes beforestimulation is required for maximum induction of a stable suppressedstate. LDL-In does not inhibit ³ H-thymidine uptake when it is added tothe cultures 18-20 hr after stimulation, suggesting that thislipoprotein influences metabolic events associated with an earlyinductive phase of lymphocyte activation.

The immunosuppressive activity of LDL-In has been studied in a number ofsystems both in vitro and in vivo. To summarize, in vitro activities ofLDL-In include suppression of: a) mitogen stimulated ³ H-thymidineuptake, Curtiss et al., J. Immunol., 116:1452, (1976), b) allogeniccell-stimulated ³ H-thymidine uptake (Curtiss et al., J. Immunol.,116:1452, (1976), Curtiss et al., J. Immunol., 118:1966, (1977)), c) theprimary generation of cytotoxic T cells (Edgington et al., RegulatoryMechanisms in Lymphocyte Activation: Proceedinqs of the EleventhLeukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York,pp. 736, (1977)), d) pokeweed mitogen stimulated immunoglobulinsynthesis (Curtiss et al., J. Clin. Invest., 63:193, (1979)), and e)B-cell Epstein Barr Virus transformation (Chisari et al., J. Clin.Invest., 68:329, (1981)). In vivo LDL-In has been shown to inhibit: a)the primary humoral immune response to sheep red blood cells (Curtiss etal., J. Immunol., 118:648, (1977), DeHeer et al., Immunopharmacology,2:9, (1979), Curtiss et al., Cell. Immunol., 49:1, (1980)), b) theprimary generation of cytotoxic T-cells (Edgington et al., RegulatoryMechanisms in Lymphocyte Activation: Proceedings of the EleventhLeukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York,pp. 736, (1977)), and c) immunologic attention of tumor growth(Edgington et al., Cancer Res., 41:3786, (1981), Edgington et al.,Dietary Fats and Health., ACOS Monograph No. 10, Perkins and Visek,eds., pp. 901, (1981)).

The effects of lipoproteins on immune cell function in vivo areexceedingly complex. A major finding of the investigation of thephysiologic implications of immunosuppression by LDL-In in vivo is thatthe observed functional outcome is strikingly dose dependent. Thisimportant concept is best illustrated by describing in more detailstudies of the effects of LDL-In on the survival of experimental animalschallenged with syngeneic tumors (Edgington et al., Cancer Res.,41:3786, (1981), Edgington et al., Dietary Fats and Health., ACOSMonograph No. 10, Perkins and Visek, eds., pp. 901, (1981)). Seeminglydivergent effects of LDL-In are observed on the growth of the syngeneicSaD2 fibrosarcoma in DBA/2 mice. The growth of 1×10⁵ viable tumor cellsin control mice without immunoprotection (i.e., 10-days priorimmunization with 10⁻⁶ irradiated tumor cells) is detectable at 25 daysand proceeds rapidly until death at about 43 days. In contrast, tumorgrowth is slower in immunoprotected mice. This tumor growth ischaracterized by a reduction in tumor mass of at least a half and nodeaths by day 60. Intravenous administration of high doses of LDL-In 24hr before immunoprotection with killed tumor cells abolishes theprotective effect of immunization. This dose corresponds to a dose thatis required to abolish both B-cell and T-cell effector cell functions.The administration of an intermediate dose of LDL-In beforeimmunoprotection with the killed tumor cells has no discernable effecton the subsequent growth of the viable tumor cell challenge. Incontrast, intravenous administration of even lower doses of LDL-In 24 hrbefore immunoprotection with killed tumor cells results in theenhancement of tumor rejection and host survival. This dose of LDL-In isconcordant with the dose required for selective inhibition of suppressorcell function in vitro (Curtiss et al., J., Clin. Invest., 63:193,(1979)). Thus, depending upon the amount of immunoregulatory lipoproteinthat a particular lymphocyte population is exposed to in vivo, verydifferent functional outcomes will result.

Further substantiation that apo E and Apo B-containing lipoproteins areimportant regulators of lymphocyte function has come from studies of theinhibitory properties of fetal cord blood plasma lipoproteins (Curtisset al., J. Immunol., 133:1379, (1984)). In these studies a directcorrelation between apo E and inhibition was established.

Cardin et al., Biochem. Biophys. Res. Comm., 154:741-745 (1988) reportedthat a polypeptide portion of apo E having an amino acid residuesequence identical to that of apo E residues 141-155 inhibits lymphocyteproliferation when coupled to bovine serum albumin (BSA). However,conspicuously absent from the study of Cardin et al. was any control forcell viability allowing for a determination of whether or not theinhibition observed was due to cytotoxicity of the peptide-BSAconjugate.

By way of further background, Dyer et al., J. Biol. Chem.,263:10965-10973 (1988) reported that isolated lipid free rat Apoinhibits androgen production by the ovarian theca and interstitial cellsinduced by the gonadotropin, luteinizing hormone (LH).

More recently, Dyer et al, J. Biol. Chem., 266:15009-15015, (1991), haveshown that only multimers, and not monomers, of the apo E polypeptidep141-155 exhibit the biological activity of apo E.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that the amino acid residue sequencecorresponding to residues 141-150 of mature apo E defines a site on apoE involved in LDL-receptor binding. Polypeptides derived from the141-150 region of apo E can mimic the biological activity of apo E whenpresent as a multimeric peptide or a self-conjugate. A multimeric apo Epolypeptide is useful for preparing antibodies, and in diagnosticmethods to monitor the amounts of apo E antigens in a vascular bodyfluid.

Thus, the present invention contemplates a polypeptide analog of apo Echaracterized by a plurality of segments each including an amino acidresidue sequence related to the sequence corresponding to residues141-150 of apo E, e.g., (LRKLRKRLLR)_(a) where a is an integer of atleast 2 indicating the number of times the sequence within parenthesisis present within the primary structure of the polypeptide.

In another embodiment, the present invention contemplates aself-conjugate of the above polypeptide, i.e., having multiplepolypeptides operatively linked to each other by other than a peptidebond between the alpha-amino group and carboxy group of contiguous aminoacid residues.

The polypeptides and conjugates of the present invention are usefulagents for modulating differentiated cell function, such as lymphocyteproliferation, ovarian androgen production, LDL binding and degradation,and the like. Therapeutic compositions containing a polypeptide orconjugate of this invention in a pharmaceutically acceptable excipient,typically in unit dose form, are also contemplated for modulatingdifferentiated cell function.

Another aspect contemplated by this invention is a compositioncomprising antibody molecules that immunoreact with a polypeptide ofthis invention and with apo E/VLDL, but do not immunoreact with thepolypeptide p93-112 or p172-182 and preferably do not immunoreact with apolypeptide containing only a monomer of p141-150.

Further contemplated is a method for detecting apo E antigens in avascular fluid sample by admixing the sample with an anti-apo E antibodyof this invention to form an immunoreaction admixture, the antibodypreferably being operatively linked to a solid support such that theimmunoreaction admixture has both a liquid phase and a solid phase. Thisimmunoreaction admixture is maintained under biological assay conditionsfor a time period sufficient to form an apo E immunoreaction product.Thereafter the amount of immunoreaction product thus formed is detected,and thereby the amount of apo E antigen present in the vascular fluidsample, is determined.

Still further contemplated is a diagnostic system, in kit form,comprising, in an amount sufficient to perform at least one assay, anantibody composition of this invention.

Also contemplated is a method for monitoring the efficacy of atherapeutic regimen for facilitating cholesterol clearance. This methodcomprises (i) determining the total amount of apo E components invascular fluid, and (ii) determining the amount of effective apo Eassociated with cholesterol-containing lipoprotein particles in vascularfluid. Thus, the total apo E receptor-competent apo E ratio isdetermined, and the resultant ratio is related to predeterminedconcentration levels that have previously been correlated to degree ofcholesterol clearance efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate that tandem apo E peptide p(141-155)₂ affectsLDL binding and degradation in a dose dependent, biphasic manner asdetermined in Example 9. In FIG. 1A, increasing concentrations ofcompetitor comprising tandem peptide p(141-155)₂, LDL, control monomerp141-155 or control p74-105 derived from apolipoprotein AI (AI 74-105)were added to cultures of THP-1 cells simultaneously with the additionof ¹²⁵ I-LDL.

Figure lB shows the specificity of tandem peptide binding to LDLreceptors. THP-1 cells were stimulated with PMA for 4 days. LDL wasacetylated, radiolabeled, and the ¹²⁵ I-aLDL was used in the bindingstudy in the presence of varying concentrations of the indicatedcompetitors After 5 hours at 37° C. the B/B_(o) ratios were analyzed andexpressed as in FIG. 1A. Neither LDL or tandem peptide inhibiteddegradation of ¹²⁵ I-aLDL (FIG. 1B), but the tandem peptide caused an80% inhibition of ¹²⁵ I-LDL degradation (FIG. 1A).

FIG. 2 illustrates that specific amino acid substitutions in the tandempeptide p(141-155)₂ alter its ability to inhibit ¹²⁵ I-LDL binding tofibroblasts when measured as described in Example 10. Each point is theaverage of 3 replicates per treatment with the SEM<10%. Shown are:native tandem peptide p(141-155)₂, and tandem peptide having thesubstitutions Lys 143→Ala; Leu 144→Pro; and Arg 150→Ala.

FIG. 3 illustrates that the trimer peptide p(141-155)₃ is a more potentinhibitor of fibroblast LDL degradation than the tandem peptidep(141-155)₂ when assayed as described in Example 11. Each point is theaverage of 5 replicates per treatment with SEM<10% and the results areexpressed as B/B_(o) as described in the descriptions of FIGS. 1A and1B. Shown are trimer peptide p(141-155)₃, tandem peptide p(141-155)₂,monomer peptide p(141-155), and a control apo AI peptide (AI 74-105).

FIG. 4 illustrates the effects on lymphocyte proliferation of monomers,dimers, trimers and tetramers of apo E (141-155) as described in Example17. (open circle=monomer; filled circle=dimer; open triangle=trimer;filled triangle=tetramer; open square=phosphate buffered saline).

FIG. 5 illustrates the effects on lymphocyte proliferation of singleamino acid substitutions in both positions of the tandem p(141-155)₂peptide. The assays were performed as in Example 2 and the substitutionswere identical to those in Example 10. (closed circle=unsubstitutedtandem p(141-155)₂ ; open square=tandem with Leu 144→Pro substitution;open triangle=tandem with Arg 150→Ala substitution; closedtriangle=tandem with Lys 143→Ala substitution).

FIG. 6 illustrates the effects on lymphocyte proliferation of variouspolypeptides derived from the sequence of apo E and described in Example19. closed circle=p(141-155)₂ ; open square=p(144-150)₂ ; opencircle=p(145-155)₂ ; closed triangle=p(141-150)₂.

FIG. 7 illustrates the effects on lymphocyte proliferation of variouspolypeptides derived from the sequence of apo E and described in Example19. open square=dimer p(141-155)₂ ; closed triangle=p(LLRK)₄ ; closedtriangle=p(LLRK)₈ ; closed square=Tris buffer control.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: The amino acid residues described herein arepreferred to be in the "L" isomeric form. However, residues in the "D"isomeric form can be substituted for any L-amino acid residue, as longas the desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. In keeping with standard polypeptidenomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations foramino acid residues are shown in the following Table of Correspondence:

                  TABLE OF CORRESPONDENCE                                         ______________________________________                                        ABBREVIATION                                                                  1-Letter    3-Letter      AMINO ACID                                          ______________________________________                                        Y           Tyr           tyrosine                                            G           Gly           glycine                                             F           Phe           phenylalanine                                       M           Met           methionine                                          A           Ala           alanine                                             S           Ser           serine                                              I           Ile           isoleucine                                          L           Leu           leucine                                             T           Thr           threonine                                           V           Val           valine                                              P           Pro           proline                                             K           Lys           lysine                                              H           His           histidine                                           Q           Gln           glutamine                                           E           Glu           glutamic acid                                       W           Trp           tryptophan                                          R           Arg           arginine                                            D           Asp           aspartic acid                                       N           Asn           asparagine                                          C           Cys           cysteine                                            X           Xaa/Xbb/Xcc   variable                                            ______________________________________                                    

It should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino acid residues.

Polypeptide: refers to a linear series of amino acid residues connectedto one another by peptide bonds between the alpha-amino group andcarboxy group of contiguous amino acid residues.

Peptide: as used herein refers to a linear series of no more than about50 amino acid residues connected one to the other as in a polypeptide.

Protein: refers to a linear series of greater than 50 amino acidresidues connected one to the other as in a polypeptide.

Synthetic peptide: refers to a chemically produced chain of amino acidresidues linked together by peptide bonds that is free of naturallyoccurring proteins and fragments thereof.

B. Apo E Polypeptide

The present invention contemplates a polypeptide capable ofsubstantially mimicking the ability of apo E to induce differentiatedcellular function, such as hepatic LDL degradation, lymphocyteproliferation, androgen secretion by ovarian theca and interstitialcells, and the like. That is, a subject polypeptide acts as an analog ofapo E at least with regard to the ability of apo E to inhibit lymphocyteproliferation and/or ovarian androgen secretion, and increase the uptakeof LDL by hepatocytes.

A subject polypeptide is further characterized by comprising a pluralityof apo E-derived segments (regions) within the polypeptide's primarystructure, each of the segments being defined by a sequence of aminoacid residues corresponding to the formula:

    Leu-Arg-Xaa-Leu-Arg-Lys-Arg-Leu-Leu-Xbb,

where Xaa is Lys or Ala and Xbb is Arg or Ala. Preferably, thepolypeptide comprises a plurality of segments, each having an amino acidresidue sequence corresponding to the formula:

    Leu-Arg-Xaa-Leu-Arg-Lys-Arg-Leu-Leu-Xbb-Asp-Ala-Asp-Asp-Leu,

where Xaa is Lys of Ala and Xbb is Arg or Ala.

In one embodiment, a segment included in a polypeptide has an amino acidresidue sequence that corresponds to the amino acid residue sequence ofapolipoprotein E. However, certain amino acid substitutions aredescribed herein whereby the polypeptide sequence can differ from an apoE sequence. Preferred polypeptides can include the substitutionsdescribed in the above formula at residues Xaa and Xbb, corresponding topositions 143 and 150, respectively, of apo E.

Thus, in one embodiment preferred polypeptides comprise a plurality ofsegments wherein at least one of the segments has an amino acid residuesequence corresponding to a formula selected from the group consistingof:

(1) Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg,

(2) Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Arg,

(3) Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Ala,

(4) Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Ala,

(5) Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg-Asp-Ala-Asp-Asp-Leu,

(6) Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Arg-Asp-Ala-Asp-Asp-Leu,

(7) Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Ala-Asp-Ala-Asp-Asp-Leu, and

(8) Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Ala-Asp-Ala-Asp-Asp-Leu.

The apo E-derived segments are capable of binding to the LDL receptorand/or LDL receptor-related protein [Herz et al., EMBO Journal,7:4119-4129 (1988)] as evidenced by the ability of the binding to becompetitively inhibited as shown herein.

The apo E-derived segments can be adjacent and/or contiguous within thepolypeptida chain, with adjacent segments being separated in the aminoacid residue sequence of the polypeptide by one or more spacing residue.Preferably, the spacing residues make up a spacing segment in the rangeof about 1 to about 20, preferably about 5 to about 15, and more usuallyabout 10, amino acid residues in length.

In addition, a subject polypeptide can contain a leader segment of 1conveniently up to about 33, such as about 11, about 18 or about 22,amino acid residues located amino-terminal to the amino-terminal apoE-derived or spacing segment.

In a similar manner, a subject polypeptide need not end with thecarboxy-terminal residue of an apo E-derived segment or spacer segment.A carboxy terminal tail segment can be present containing 1 convenientlyup to about 33, such about 11, about 18 or about 22, amino acidresidues.

Preferred polypeptides of the present invention can be defined byformula I:

    B-(X.sub.n -Leu-Arg-Xaa-Leu-Arg-Lys-Arg-Leu-Leu-Xbb-Z.sub.m).sub.a -J.

In the above formula, B is an amino-terminal NH₂ group or a previouslydiscussed leader segment; J is a carboxy-terminal COOH group or apreviously discussed tail segment; X and Z are first and second,respectively, spacing segments whose amino acid residue sequences can bethe same or different; n is either 1 or 0 such that when n is 1, X ispresent, and when n is 0, X is not present; m is either 1 or 0 such thatwhen m is 1, Z is present, and when m is 0, Z is not present; where Xaais Lys of Ala and Xbb is Arg or Ala; and a is an integer from 2 to about10, more preferably 2 to about 5 and usually 2 to 3, indicating thenumber of times the amino acid residue sequence in parenthesis ispresent (repeated) in the polypeptide primary structure. Preferably, thesequence in parenthesis corresponds in its entirety, and preferably isidentical to, a portion of the amino acid residue sequence of apo E.

In preferred polypeptides according to formula I, n is 0 and m is 0. Inanother embodiment, B is an amino-terminal NH₂ group, J is acarboxy-terminal COOH group, n is 0, and m is 0. More preferably, B isan amino-terminal NH₂ group, J is a carboxy-terminal COOH group, n is 0,m is 0, and a is 2, 3 or 4.

Based on the disclosures herein, it has also been discovered that thefunctional element of an apo E analog polypeptide of this invention thatconveys the properties of an Apo E mimic involves alternatinghydrophobic and basic amino acid residues to mimic the repeating element(segment) of a polypeptide of the invention. This embodiment is based ofthe discovery herein that the active LDL-receptor binding moiety of ApoE resides in the p141-150 region, and the further discovery that analogswith tandem repeats containing two basic residues such as arginine andlysine and hydrophobic spacers such as leucine-leucine can form anactive analog.

Thus in a related embodiment, the present invention contemplates a Apo Epolypeptide analog having the properties of a subject polypeptide whichcan be represented according to formula II:

    (Z.sub.n B.sub.m).sub.a,

where Z is a hydrophobic amino acid residue, B is a basic amino acidresidue, n is 2 to 100, preferably 2 to 20, and more preferably 2 to 3,m is 2 to 4, preferably 2, and a is 2 to 100, preferably 2 to 50, andmore preferably 2 to 8. In preferred embodiments, Z is isoleucine,leucine or valine. In preferred embodiments, B is arginine, lysine orhistidine, and more preferably is arginine or lysine.

In this embodiment, exemplary and preferred polypeptide analogs have theformula p(LLRK)₄ and p(LLRK)₈.

Particularly preferred polypeptides of this invention are those whoseformulas are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Designation.sup.1                                                                     Amino Acid Residue Sequences                                          __________________________________________________________________________    p(141-150).sub.2                                                                      LRKLRKRLLRLRKLRKRLLR                                                  p(141-155).sub.2                                                                      LRKLRKRLLRDADDLLRKLRKRLLRDADD                                         p(K.sub.143 --A).sub.2                                                                LRALRKRLLRDADDLLRALRKRLLRDADD                                         p(K.sub.150 --A).sub.2                                                                LRKLRKRLLADADDLLRKLRKRLLADADD                                         p(141-155).sub.3                                                                      LRKLRKRLLRDADDLLRKLRKRLLRDADD-                                                LRKLRKRLLRDADDL                                                       p(141-155).sub.4                                                                      LRKLRKRLLRDADDLLRKLRKRLLRDADD-                                                LRKLRKRLLRDADDLLRKLRKRLLRDADD                                         p(129-163).sub.2                                                                      STEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQ-                                          STEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQ                                   p(LLRK).sub.4                                                                         LLRKLLRKLLRKLLRK                                                      p(LLRK).sub.8                                                                         LLRKLLRKLLRKLLRKLLRKLLRKLLRKLLRK                                      __________________________________________________________________________     .sup.1 The designation for each peptide indicates the position within the     amino acid residue sequence of the mature Apo E protein to which the          peptide sequence corresponds, i.e., is derived from, or indicates a           formula representative of the sequence shown. p(K.sub.143 --A).sub.2          refers to p(141-155).sub.2 except that an alanine (A) is substituted for      lysine (K) at position 143 in both copies of the 141-155 segment, and         p(K.sub.150 --A).sub.2 refers to p(141-155).sub.2 except that an alanine      (A) is substituted for a arginine (R) at position 150 in both copies of       the 141-155 segment.                                                     

It should be noted that p(129-163)₂ contains a 12 residue leader segmentSTEELRVRLASH (residues 1-12), a 20 residue spacing segmentQKRLAVYQSTEELR-VRLASH (residues 28-47) and an 8 residue tail segmentQKRLAVYQ (residues 63-70). The designation p(141-155)₂ defines a tandemapo E peptide which contains two adjacent sequences of the p141-155segment. An additional preferred polypeptide is a "trimer" containingthree adjacent sequences of the p141-155 segment, designatedp(141-155)₃. Preferred also are self-conjugates of the tandem apo Epeptides designated p(141-155)₂ - p(141-155)₂ and timer apo E peptidesdesignated p(141-155)₃ - p(141-155)₃. The designation p(141-150)₂defines a tandem apo E peptide which contains two adjacent sequences ofthe p141-150 segment. Also shown in Table 1 are a tetramer of p(141-155)and two dimers of p(141-155) which contain substitutions at positions143 or 150 of apo E.

A preferred polypeptide of this invention in one embodiment has asequence corresponding to the designation selected from the groupconsisting of p(141-150)₂, p(141-155)₂, p(141-155)₃, p(141-155)₄, p(K₁₄₃-A)₂, p(K₁₅₀ -A)₂ and p(129-163)₂, where the sequences are shown inTable 1.

A subject polypeptide typically contains a total of about 30 to about450 amino acid residues, preferably about 60 to about 120 residues.Typically, a subject polypeptide contains no more than about 100,preferably no more than about 70 and usually no more than about 30 or 40amino acid residues in its primary sequence.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is capable of inducing differentiatedcellular function in a manner corresponding to that of apo E. Therefore,a present polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use.

The term "analog" includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the ability to mimicapo E as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase "conservative substitution" also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

"Chemical derivative" refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

The term "fragment" refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

A subject polypeptide can be prepared using recombinant nucleic acidmethodologies well known in the art. For instance, DNA sequences usefulin producing a subject polypeptide are described in Paik et al., Proc.Natl. Acad. Sci. USA, 82:3445-3449, (1985); McLean et al., J. Biol.Chem., 259:6498-6504, (1984); and Rall et al., J. Biol. Chem.,257:4171-4178, (1982). A DNA segment coding for a polypeptide of thisinvention can be synthesized by chemical techniques, for example thephosphotriester method of Matteucci et al., J. Am. Chem. Soc,, 103:3185,(1981). The DNA segment can then be ligated into an expression vector,and a host transformed therewith can be used to produce the polypeptide.See, for example, Current Protocols In Molecular Biology, Ausubel etal., eds., John Willey & Sons, New York, N.Y.; U.S. Pat. Nos. 4,237,224and 4,356,270.

The recombinant expression vectors capable of expressing a subjectpolypeptide and methods of their use for producing a subject polypeptideare contemplated as part of the present invention.

A subject polypeptide can also be prepared using the solid-phasesynthetic technique initially described by Merrifield, in J. Am. Chem.Soc., 85:2149-2154 (1963). Other polypeptide synthesis techniques may befound, for example, in M. Bodanszky et al., Peptide Synthesis, JohnWiley & Sons, 2d Ed., (1976) as well as in other reference works knownto those skilled in the art. A summary of polypeptide synthesistechniques may be found in J. Stuart and J.D. Young, Solid Phase PeptideSynthesis, Pierce Chemical Company, Rockford, Ill., 3d Ed., Neurath, H.et al., Eds., p. 104-237, Academic Press, New York, N.Y. (1976).Appropriate protective groups for use in such syntheses will be found inthe above texts as well as in J. F. W. McOmie, Protective Groups inOrganic Chemistry, Plenum Press, New York, N.Y. (1973).

In general, those synthetic methods comprise the sequential addition ofone or more amino acid residues or suitably protected amino acidresidues to a growing polypeptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amid linkage with the residue already attached to the solid support.The protecting group of the amino or carboxyl group is then removed fromthis newly added amino acid residue, and the next amino acid (suitablyprotected) is then added, and so forth. After all the desired aminoacids have been linked in the proper sequence any remaining terminal andside group protecting groups (and solid support) are removedsequentially or concurrently, to provide the final polypeptide.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

C. Conjugates

The present invention further contemplates an apo E analog in the formof a polypeptide conjugate comprised of a plurality of polypeptidesoperatively linked, by other than a peptide bond between the alpha-aminogroup and carboxy group of contiguous amino acid residues, where atleast two of the linked polypeptides have an amino acid residue sequencecorresponding to that represented by the formula:

    B-(X.sub.n -Leu-Arg-Xaa-Leu-Arg-Lys-Arg-Leu-Leu-Xbb-Z.sub.m).sub.a -J,

wherein B, X, Z, J, Xaa, Xbb, n, m and a are defined as previouslydiscussed except that a can also be the integer 1.

Preferred self-conjugates are p141-150 linked to p141-150, designated(p141-150)-(p141-150), and similar conjugates of p141-155, and otherpolypeptides of the invention.

Additional conjugates contemplated comprise a polypeptide according toformula II described herein in the operative linkage of a conjugate.

In preferred embodiments, a conjugate of this invention has a molecularweight of less than about 40,000 daltons, preferably less than about20,000 daltons, and more preferably less than about 10,000 daltons.Typically, a subject conjugate has a molecular weight of no more thanabout 15,000 daltons, preferably no more than about 8,000 daltons, andusually no more than about 4,000 daltons. Preferably, the conjugate isdimeric or trimeric, i.e., consists essentially of two or threepolypeptide chains, respectively.

A polypeptide conjugate of this invention is further characterized byits ability to substantially mimic apo E's ability to inducedifferentiated cellular function, such as lymphocyte proliferation,ovarian androgen secretion, and the like. The subject conjugates arealso substantially free of toxicity toward lymphocytes andandrogen-producing ovarian (theca/interstitial) cells at concentrationsof about 20 micrograms per milliliter (μg/ml).

The techniques of polypeptide conjugation or coupling through activatedfunctional groups presently known in the art are particularlyapplicable. See, for example, Avrameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. Nos. 4,493,795, 3,791,932 and3,839,153. In addition, a site directed coupling reaction can be carriedout so that any loss of activity due to polypeptide orientation aftercoupling can be minimized. See, for example, Rodwell et al., Biotech.,3:889-894 (1985), and U.S. Pat. No. 4,671,958.

One or more additional amino acid residues may be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto form a conjugate. Cysteine residues, usually added at thecarboxy-terminus of the polypeptide, have been found to be particularlyuseful for forming conjugates via disulfide bonds, but other methodswell-known in the art for preparing conjugates may be used.

D. Compositions for Modulatinq Hepatic LDL Degradation

In view of the ability of the polypeptides and conjugates of the presentinvention to bind the LDL receptor present on hepatocytes, the presentinvention contemplates a composition for modulating hepatic uptake ofLDL. The composition comprises an LDL receptor-binding moietyoperatively linked to an LDL binding moiety. The LDL receptor-bindingmoiety comprises a polypeptide and/or conjugate of the presentinvention. A preferred LDL receptor-binding moiety comprises thepolypeptide segment according to formula I or formula II describedearlier. Particularly preferred LDL receptor-binding moieties have anamino acid residue sequence shown in Table 1.

The LDL receptor-binding moiety can be operatively linked to the LDLbinding moiety by a peptide bond or through a covalent bond that is nota peptide bond between the alpha-amino group and carboxyl group ofcontinuous amino acid residues.

An LDL binding moiety can be an anti-LDL antibody molecule orimmunologically active fragment thereof. Exemplary anti-LDL-antibodymolecules are produced by hybridomas HB8746 and HB8742, which have beendeposited with the American Tissue Culture Collection (ATCC; Rockville,Md.), both of which produce anti-apo B-100 antibody molecules. The LDLreceptor-binding polypeptide and/or conjugate of this invention can bechemically coupled as described hereinbefore to the anti-LDL antibodymolecule. Alternatively, a polypeptide of this invention can beincorporated into the primary amino acid residue sequence of theantibody molecule by recombinant DNA techniques. Typically, the LDLreceptor-binding polypeptide will be incorporated into or substitutedfor a portion of one of the antibody molecule's constant domains. SeeU.S. Pat. Nos. 4,816,567, 4,816,397 and 4,647,334.

In preferred embodiments, the LDL binding moiety is a lipophilic(hydrophobic) sequence of amino acid residues. More preferably, the LDLbinding moiety is a polypeptide segment having an amino acid residuesequence capable of forming an amphipathic helix.

Of course, when the means for operatively linking the LDL receptormoiety and LDL binding moiety is other than a peptide bond, the linkingtypically occurs between amino acid residue chains on residues at ornear the carboxy-and/or amino-terminus of the respective moieties so asto preserve their activities.

Preferred helical amphipathic polypeptide segments of this invention,whether incorporated into the composition by a peptidic or non-peptidicbond, include those having an amino acid residue sequence correspondingto that of an apolipoprotein, such as apo B-100, apo B-48, apo C-I, apoC-II, apo C-III, apo A-I, apo A-II, apo D, apo E and the like. See,Fitch, Genetics, 86:623-644 (1977); Segrest et al., Biopolymers,16:2053-2065 (1977); and Chan, Klin Wochenscher, 67:225-237 (1989). Byusing a helical amphipathic polypeptide segment with amino acid residuesequence derived from apo B-100 or apo B-48, the polypeptide can bepreferentially targeted to LDL as opposed to other lipoprotein species.

The amphipathic helix is characterized by a spacial segregation ofhydrophobic and hydrophilic amino acid residues on opposite faces of thehelix. The clustered nonpolar residues can then intercalate into lipidparticles such as LDL. In addition to this hydrophobic interaction,there may also be specific charge interactions between lipid andpeptide. For example, it has been demonstrated that an 18-residuepeptide can bind to phospholipid if it has positively charged residuesat the hydrophobic-hydrophilic interface of an amphipathic helix andnegatively charged residues opposite the hydrophobic face of the helix.See Epand et al., J. Biol. Chem., 264:4628-4635 (1989) .

A particularly preferred helical amphipathic polypeptide segment usefulin binding the apo E-derived polypeptide segment LDL receptor-bindingmoiety to LDL has an amino acid residue sequence corresponding to theformula:

EWLKAFYEKVLEKLKELF.

In preferred embodiments, the composition is a polypeptide according toformula I wherein B and/or J is a helical amphipathic polypeptidesegment as described above. One preferred polypeptide of this type hasan amino acid residue sequence corresponding to the formula:

LRKLRKRLLRDADDLLRKLRKRLLRDADDLEWLKAFYEKVLEKLKELF.

In preferred embodiments, the subject polypeptide or conjugate isdispersed in a carrier, such as a phospholipid. More preferably, thesubject polypeptide or conjugate is removably inserted in a liposome,i.e., it is incorporated (anchored) into the liposome bilayer via theLDL binding moiety. See, for example, Gregoriadis, Trends in Biotecht,3:235-241 (1985) and Eriksson et al., pp 141-156 in Liposome TechnologyVol. II, ed G. Gregoriadis CRC Press, Boca Raton, Fla.

E. Therapeutic Methods

The polypeptides, conjugates and compositions contemplated by thepresent invention are useful as agents for modulating those physiologicevents induced by native apo E, such as immune response, steroidogenesisand/or enhance hepatic LDL-binding. For instance, a polypeptide and/orconjugate of this invention can be used as an immunosuppressive agent toinhibit the proliferation of lymphocytes or as an agent to inhibitovarian androgen production. The polypeptide and/or conjugate isadministered to the animal, such as a human, in need of such treatment,in a predetermined amount calculated to achieve the desired effect,i.e., in a therapeutically effective amount.

Typically, a higher order of multimer was shown herein to produce apolypeptide of higher specific activity. Thus, the effectiveconcentration will depend on the specific polypeptide being utilized.

Therefore, when used as an immunosuppressive agent for inhibitinglymphocyte proliferation, such as in a patient displaying the symptomsof an autoimmune disease, the tandem polypeptide and/or conjugate isadministered in an amount sufficient to achieve a plasma concentrationof at least about 0.8 μg/ml, preferably at least about 1.0 to 2.0 μg/mlon up to about 30 to 50 μg/ml depending on the specific polypeptidebeing utilized. Stated in different terms, an inhibitory amount is anamount sufficient to produce a plasma concentration of from at leastabout 1-5 μM to about 10-50 μM, depending upon the compound being used.

In some cases, it is desirable to apply the subject polypeptide and/orconjugate locally as an immunosuppressive agent. For instance, about 10μg to about 1 mg can be applied by injection into an arthritic joint(e.g., into the synovial fluid of the joint) to suppress inflammation.

When used as an agent for inhibiting ovarian androgen production, suchas in females having polycystic ovaries, a polypeptide and/or conjugateof this invention is administered in an amount sufficient to achieve aplasma concentration of at least about a 2 μg/ml, preferably at leastabout 5 μg/ml, and more preferably about 10 μg/ml (i.e., about 1-50 uM).

In contrast, the polypeptides and conjugates were observed to enhancelymphocyte proliferation and enhance androgen synthesis when used atlower concentrations than for inhibition. The mechanism for this"biphasic" drug response is not fully understood, but is reproduciblyobserved when the therapeutic compositions were administered atconcentrations lower than inhibitory concentrations.

When used to enhance lymphocyte proliferation or androgen synthesis, thetandem peptide or its conjugate is given in an amount sufficient toachieve a plasma concentration of from about 0.1 μg/ml to about 5 μg/ml,and in some situations as high as 1 μg/ml, (i.e., about 0.1-1 uMdepending on the specific compound being used).

A third biological response observed by the polypeptides or selfconjugates of this invention was to modulate hepatic LDL binding anduptake, as in subjects with hyper-cholesterolemia.

In patients where it is desirable to enhance LDL binding and uptake,therefor, the composition is administered in an amount sufficient toachieve a tandem peptide or conjugate plasma concentration of about 0.1to 1.0 uM

In contrast, in patients where it is desirable to inhibit LDL bindingand uptake, the composition is administered in an amount sufficient toachieve a tandem peptide or conjugate plasma concentration of at leastabout 1.0 uM, preferably about 5 to 50 uM, depending upon the specificpolypeptide or conjugate being tested.

Precise effective concentrations of a polypeptide or conjugate of thisinvention depend on the specific compound, and the desired effect, asshown herein. Effective concentrations of a particular composition foruse as an inhibitor or enhancer in a apo E mediated process as describedherein can readily be determined by an assay for activity as describedherein.

The preparation of therapeutic compositions which contain polypeptidesas active ingredients is well understood in the art. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired,the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

A polypeptide can be formulated into the therapeutic composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids,or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The therapeutic polypeptide-containing compositions are conventionallyadministered intravenously or at the site of autoimmune-inducedinflammation, as by injection of a unit dose, for example. The term"unit dose" when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition of lymphoproliferation or androgen production desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are peculiar to each individual.However, suitable dosage ranges for systemic application are of theorder of 0.01 to 10, preferably one to several, milligrams of activeingredient per kilogram bodyweight of individual per day and depend onthe route of administration that result in the plasma concentrationsrecited earlier. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain the earlier recited plasmaconcentrations in the blood are contemplated.

F. Antibodies and Monoclonal Antibodies

The term "antibody" in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active.portions of immunoglobulin molecules,i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules for use in the diagnostic methods andsystems of the present invention are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab' F(ab')₂ and F(v)

Fab and F(ab')₂ portions of antibodies are prepared by the proteolyticreaction of papain and pepsin, respectively, on substantially intactantibodies by methods that are well known. See, for example, U.S. Pat.No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody portions arealso well known and are produced from F(ab')₂ portions followed byreduction of the disulfide bonds linking the two heavy reduction of thedisulfide bonds linking the two heavy chain portions as withmercaptoethanol, and followed by alkylation of the resulting proteinmercaptan with a reagent such as iodoacetamide. An antibody containingintact antibody molecules are preferred, and are utilized asillustrative herein.

An antibody of the present invention, i.e., an anti-apo E antibody, inone embodiment is characterized as being capable of immunoreacting withapo E present on cholesterol containing-lipoprotein particles such asLDL, VLDL and the like. Apo E in association with VLDL is referred to asapo E/VLDL. An anti-apo E antibody of this embodiment is furthercharacterized as immunoreacting with the polypeptide of this invention,for example a polypeptide comprising a plurality of segments having theformula p(141-150), p(141-150)₂, p(141-150)₃, p(141-150)₄, orself-conjugates of p(141-150)₂ or p(141-150)₃, or with a polypeptideaccording to formula I of II.

In a preferred embodiment, an anti-apo E antibody is characterized asbeing substantially free of antibody molecules that immunoreact with thepolypeptide LSKELQAAQARLGADMEDVR corresponding to residues 93-112 ofmature apo E and designated p93-112, or with the polypeptide RGLSAIRERLcorresponding to residues 172-182 of mature apo E and designatedp172-182.

Particularly preferred are antibody molecules that do not immunoreactwith a polypeptide containing only a monomer of the apo E polypeptidep141-150 or p141∝155. Apo E polypeptides that contain only a singlep141-150 or p141-155 do not have the capacity to mimic the LDLreceptor-binding capacity of apo E that results in enhanced hepaticuptake and degradation of apo E-containing lipoprotein particles asdisclosed herein. Thus, such an antibody is immunospecific for antigenscapable of mimicking a LDL-receptor binding moiety, and does notimmunoreact with monomers.

Polypeptides that mimic the LDL receptor binding function of apo E arereferred to as "receptor-competent" apo E polypeptides. Anti-apo Eantibody molecules that do not immunoreact with monomeric p141-150 orp141-155 thus have immunospecificity for receptor-competent apo Epolypeptides, and have immunospecificity for apo E apolipoprotein thatis in a receptor-competent form.

Anti-apo E antibody molecules that are specific for receptor-competentapo E or apo E polypeptides have a particular utility in immunoassays,namely, to monitor the fate of therapeutically administered apo Epolypeptides during the course of therapeutic regimens as disclosedherein, or to detect the levels of receptor-competent apo E in avascular body fluid sample.

Antibody immunoreactivity with apo E-containing antigens can be measuredby a variety of immunological assays known in the art. Exemplaryimmunoreaction of an anti-apo E antibody of this invention by directbinding with apo E/VLDL or with apo E polypeptides can be assayed atleast by the methods described in Example 14.

An antibody of the present invention is typically produced by immunizinga mammal with an inoculum containing an apo E polypeptide of thisinvention, such as a self-conjugate of a polypeptide according toformula I or II, or with a polypeptide according to formula I or II, orwith a polypeptide shown in Table 1, and thereby induce in the mammalantibody molecules having immunospecificity for an apo E polypeptide ofthis invention or for LDL receptor competent apo E. Alternatively, apoE/LDL or apo E/VLDL can be used as the source of immunizing apo Eantigen.

Exemplary are the production methods for preparing a polyclonal anti-apoE polypeptide antisera described in Example 8. The antibody moleculesare then collected from the mammal and isolated to the extent desired bywell known techniques such as, for example, by using DEAE Sephadex toobtain the IgG fraction.

To enhance the antibody specificity, antibodies that are purified byimmunoaffinity chromatography using solid phase-affixed immunizingpolypeptide are preferred. The antibody is contacted with the solidphase-affixed immunizing polypeptide for a period of time sufficient forthe polypeptide to immunoreact with the antibody molecules to form asolid phase-affixed immunocomplex. The bound antibodies are separatedfrom the complex by standard techniques.

In a related method to produce an anti-apo E antibody that does notsubstantially immunoreact with monomer, for example p141-150, theprepared anti-apo E antibody can be contacted with solid-phase monomericp141-150 so as to allow antibodies that immunoreact with monomericp141-150 to complex in the solid phase, and the liquid-phase antibody isthen collected to form anti-apo E antibody that is specific forreceptor-competent apo E polypeptides.

The antibody so produced can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect apo E or apo Epolypeptide present in a body sample.

The word "inoculum" in its various grammatical forms is used herein todescribe a composition containing a apo E polypeptide of this inventionas an active ingredient used for the preparation of antibodiesimmunoreactive with an apo E polypeptide. When a polypeptide is used inan inoculum to induce antibodies it is to be understood that thepolypeptide can be used in various embodiments, e.g., alone or linked toa carrier as a conjugate, or as a polypeptide polymer. However, for easeof expression and in context of a polypeptide inoculum, the variousembodiments of the polypeptides of this invention are collectivelyreferred to herein by the term "polypeptide", and its variousgrammatical forms.

For a polypeptide that contains fewer than about 35 amino acid residues,it is preferable to use the peptide bound to a carrier for the purposeof inducing the production of antibodies.

One or more additional amino acid residues can be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto a carrier. Cysteine residues added at the amino- or carboxy-terminiof the polypeptide have been found to be particularly useful for formingconjugates via disulfide bonds. However, other methods well known in theart for preparing conjugates can also be used. Exemplary additionallinking procedures include the use of Michael addition reactionproducts, dialdehydes such as glutaraldehyde, Klipstein, et al., J.Infect. Dis., 147:318-326 (1983) and the like, or the use ofcarbodiimide technology as in the use of a water-soluble carbodiimide toform amide links to the carrier. For a review of protein conjugation orcoupling through activated functional groups, see Avrameas, et al.,Scand. J. Immunol., 1:7-23 (1978).

Useful carriers are well known in the art, and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin(BSA) or human serum albumin (HSA), red blood cells such as sheeperythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyaminoacids such as poly (D-lysine; D-glutamic acid), and the like.

The choice of carrier is more dependent upon the ultimate use of theinoculum and is based upon criteria not particularly involved in thepresent invention. For example, acarrier that does not generate anuntoward reaction in the particular animal to be inoculated should beselected.

The present inoculum contains an effective, immunogenic amount of apolypeptide of this invention, typically as a conjugate linked to acarrier. The effective amount of polypeptide per unit dose sufficient toinduce an immune response to the immunizing polypeptide depends, amongother things, on the species of animal inoculated, the body weight ofthe animal and the chosen inoculation regimen as is well known in theart. Inocula typically contain polypeptide concentrations of about 10micrograms to about 500 milligrams per inoculation (dose), preferablyabout 50 micrograms to about 50 milligrams per dose.

The term "unit dose" as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for animals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired immunogenic effect in association with the requireddiluent; i.e., carrier, or vehicle. The specifications for the novelunit dose of an inoculum of this invention are dictated by and aredirectly dependent on (a) the unique characteristics of the activematerial and the particular immunologic effect to be achieved, and (b)the limitations inherent in the art of compounding such active materialfor immunologic use in animals, as disclosed in detail herein, thesebeing features of the present invention.

Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent such as water, saline orphosphate-buffered saline to form an aqueous composition.

Inocula can also include an adjuvant as part of the diluent. Adjuvantssuch as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum are materials well known in the art, and are availablecommercially from several sources.

The techniques of polypeptide conjugation or coupling through activatedfunctional groups presently known in the art are particularlyapplicable. See, for example, Avrameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. Nos. 4,493,795, 3,791,932 and3,839,153. In addition, a site directed coupling reaction can be carriedout so that any loss of activity due to polypeptide orientation aftercoupling can be minimized. See, for example, Rodwell et al., Biotech.,3:889-894 (1985), and U.S. Pat. No. 4,671,958.

One or more additional amino acid residues may be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto form a conjugate. Cysteine residues, usually added at thecarboxy-terminus of the polypeptide, have been found to be particularlyuseful for forming conjugates via disulfide bonds, but other methodswell-known in the art for preparing conjugates may be used.

A particularly preferred anti-apo E antibody is a monoclonal antibody.

The phrase "monoclonal antibody" in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

Preferred anti-apo E monoclonal antibodies are prepared as disclosedherein.

Additional monoclonal antibodies useful for practicing the diagnosticmethods of this invention are those which immunoreact with LDL, VLDL,HDL, and the like lipoprotein particles. Particularly preferred areanti-apo B-100 antibodies.

An exemplary hybridoma that secretes monoclonal antibody molecules thatimmunoreact with apo B-100 has been described previously in U.S. Pat.No. 4,677,057, which is incorporated herein by reference, and themonoclonal antibody molecules secreted by the hybridoma is referred toherein as MB47. The MB47 monoclonal antibody immunoreacts with more thanabout 90 percent of ¹²⁵ I-LDL, and with a distinct and separateconserved antigenic determinant on apo B-100. As pointed out in Young etal. (1986) Clin. Chem., 32/8:1484-1490, MB47 reacts only with apo B-100.

The above hybridoma was deposited with the American Type CultureCollection (ATCC), Rockville, Md., in accordance with the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure, on Mar. 6, 1985, and was assignedthe designation HB 8746.

The above deposit was made in compliance with the Budapest Treatyrequirements that the duration of the deposit or for 5 years after thelast request for the deposit at the depository or for the enforceablelife of a U.S. patent that matures from this application, whichever islonger. The hybridoma is replenished should it become non-viable at thedepository, and is made available to the public by the ATCC upon theissuance of a patent from this application.

G. Preparation of Monoclonal Antibodies

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) butone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature, 256:495-497 (1975), which description isincorporated by reference. The hybridoma supernates so prepared can bescreened for the presence of antibody molecules that immunoreact with anapo E polypeptide of this invention.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with an apo E polypeptide of this invention, or with anative apo E molecule, such as is present in an apo E-containinglipoprotein particle. The polypeptide-induced hybridoma technology isdescribed by Niman, et al., Proc. Natl. Sci., U.S.A., 80:4949-4953(1983), which description is incorporated herein by reference.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 G1X⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention arethen identified by screening for the immunospecificities of an anti-apoE antibody as disclosed herein, for example, the enzyme linkedimmunosorbent assay (ELISA) described in Example 16, or using the solidphase radioimmunoassay (SPRIA) described in Example 14.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate immunospecificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice, and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco, et al., Virol.,8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci., 86:5728-5732(1989); and Huse, et al., Science, 246:1275-1281 (1989).

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention.

The monoclonal antibody produced by the above method can be used, forexample, in diagnostic modalities disclosed herein where formation of anapo E-containing immunoreaction product is desired.

Hybridoma HB 8746 produces MB47 monoclonal antibody molecules and wasformed by fusing splenocytes of mice immunized with LDL andP3X63Ag8.653.1 myeloma cells. Detailed preparation of the MB47monoclonal antibody and hybridoma was reported by Young, et al. (1986)Arteriosclerosis, 6:178-188.

H. Assay Methods

Useful solid and liquid phase assay methods are discussed herein.However, the invention is not so limited. Further, while theparticularly described assay methods utilize an enzyme-linked indicatormeans, the present invention is not specifically limited to such assays.Additional assay methods are described hereinbelow with particularemphasis on solid phase immunoassay methods.

Those skilled in the art will understand that there are numerous methodsof solid phase immunoassays that may be utilized herein. Exemplary,useful solid phase assays include enzyme multiplied immunoassaytechniques (EMIT) and fluorescence immune assays (FIA), in addition tothe specifically discussed RIA, solid phase radioimmunoassay (SPRIA) ofExample 8B, and ELISA. However, any method that results in a signalimparted by the reaction of apo E with an antibody of this invention isconsidered. Each of those assay methods can employ single or doubleantibody techniques in which an indicating means is utilized to signalthe immunoreaction, and thereby the binding of an apo E that is to beassayed with a receptor of this invention. Exemplary techniques can befound explained in Maggio, Enzyme Immunoassay, CRC Press, Cleveland,Ohio (1981); and in Goldman, Fluorescent Antibody Methods, AcademicPress, New York, N.Y. (1980).

A vascular fluid sample is utilized in this assay method. The sample canbe either serum or plasma. Results obtained using both compositions havepreviously been found to be statistically indistinguishable. Regardlessof whether serum or plasma is used, the vascular fluid sample ispreferably obtained from persons who have fasted for at least abouttwelve hours as is known in the art. Such a blood sample is referred toas a "fasting" sample.

One contemplated assay method determining the presence, and preferablythe amount of a LDL-receptor competent apo E in a vascular fluid sample.This method includes the following steps:

(a) A vascular fluid sample is admixed with an anti-apo E antibodycomposition of this invention to form an immunoreaction admixture. Theantibody molecules in the composition preferably are operatively linkedto a solid support such that the immunoreaction admixture has both aliquid phase and a solid phase. These antibody molecules immunoreactwith:

(i) a polypeptide of this invention, such as one according to formula I,formula II or a polypeptide according to Table 1, and

(ii) apo E/VLDL, but do not immunoreact with a polypeptide correspondingto one of the formulas p93-112 and p172-182, and preferably do notimmunoreact with a polypeptide containing only a monomer of thepolypeptide p141-150, p141-150 or p(LLRK).

(b) The immunoreaction admixture is maintained under biological assayconditions for a time period sufficient to form an apo E-containingimmunoreaction product in the solid phase.

(c) The presence, and preferably the amount of immunoreaction productformed in step (b) and thereby apo E antigen in the vascular fluidsample is then determined.

Biological assay conditions are those conditions that are able tosustain the biological activity of the immunochemical reagents of thisinvention and the antigen sought to be assayed. Those conditions includea temperature range of about 4 degrees C. to about 45 degrees C., a pHvalue range of about 5 to about 9 and an ionic strength varying fromthat of distilled water to that of about one molar sodium chloride.Methods for optimizing such conditions are well known in the art.

In a preferred embodiment of the above method the amount ofimmunoreaction product is determined according to step (c) by (i)admixing a labeled specific binding agent capable of binding an apoE-containing particle with the apo E-containing immunoreaction productto form a labeling reaction admixture, (ii) maintaining the labelingreaction admixture under biological assay conditions for a time periodsufficient for the labeled specific binding agent to bind the apoE-containing immunoreaction product to form a labeled complex, and (iii)detecting the amount of any labeled complex formed, and therebydetecting the amount of apo E-containing immunoreaction product.

In a particularly preferred embodiment, the labeled specific agent is amonoclonal anti-B-100 antibody produced by the hybridoma having ATCCdesignation HB 8742.

Another contemplated assay of this invention is a competition assaymethod for detecting the presence, and preferably the amount, of an apoE antigen in a vascular fluid sample. This assay method comprises thefollowing steps:

(a) A vascular fluid sample is admixed with a solid phase-bound apo Epolypeptide of this invention to form a first solid-liquid phaseadmixture.

(b) An antibody composition, containing a limiting amount of anti-apo Epolypeptide antibody molecules that immunoreact with the polypeptide inthe solid phase, and immunoreact with apo E/VLDL, but do not immunoreactwith a polypeptide corresponding to one of the formulas p93-119 andp72-182, and preferably do not immunoreact with a polypeptide containingonly a monomer of the polypeptide p141-150 or p141-155, is admixed withthe first admixture to form a second admixture.

(c) This second admixture is maintained under biological assayconditions for a period of time sufficient to form an apo E-containingimmunoreaction product in the solid phase.

(d) The amount of immunoreaction product present in the solid phaseformed in step (c) is determined, and thereby the amount of apo E in thevascular fluid sample.

In an alternative embodiment, the solid phase-bound apo E polypeptide isreplaced by an apo E-containing lipoprotein, such as a VLDL particle.

In a more preferred embodiment, the solid phase-bound apo E polypeptideor apo E containing component is the above-described receptor-competentpolypeptide analog, and the anti-apo E analog antibody in step (b) isoperatively linked to an enzyme indicating means, and the product formedin step (c) is a labeled immunoreaction product.

Insofar as the present diagnostic methods can be used to monitor thefate of therapeutically administered apo E polypeptides as disclosedherein, it is understood that the disclosed methods for detecting an apoE antigen can readily be applied to monitor an analog of an apo Eantigen, namely, an apo E polypeptide. For this reason, the phrase "apoE antigen" refers to antigenic molecules that immunoreact with theantibodies of the present invention, whether the antigenic molecules arenative apo E, apo E polypeptides, or combinations of both analog andnative protein.

In embodiments for following the fate of a therapeutically administeredapo E polypeptide, it is to be understood that native apo E and apo Epolypeptide may be indistinguishable in a vascular fluid sample whereboth components immunoreact with the anti-apo E antibodies. Therefore,it is useful and preferred to measure vascular levels of apo E antigenin a patient prior to the administration of a therapeutic composition toestablish a baseline of apo E antigen in the patient. At predeterminedtime intervals after administration of therapeutic peptide, thepatient's blood is then sampled and the levels of apo E antigen areagain measured to determine the effect and fate of the therapeuticcomposition on circulating apo E antigen.

Also contemplated is a related method for detecting apo E present inlipoprotein particles in a vascular fluid sample. The fluid sample isadmixed with a solid-phase anti-B-100 antibody, i.e., operatively linkedto a solid matrix, to form a liquid-solid phase immunoreactionadmixture. The admixture is maintained under biological assay conditionsfor a time period sufficient to form an immunoreaction product in thesolid phase. The immunoreaction product, containing lipoproteinparticles with apo B-100, is then admixed with an anti-apo E antibody ofthis invention to form a second liquid-solid phase immunoreactionadmixture. The second admixture is maintained as before to allow theanti-apo E antibodies to immunoreact with the solid-phase lipoproteinparticles and form a second solidphase immunoreaction product. Theresulting second product is detected to indicate the presence of apo Ein the vascular fluid.

In preferred embodiments, the anti-apo E is labeled, and the secondproduct is thereby detected by detecting the presence of label in thesolid phase. More preferably, the anti-apo E antibody has the capacityto bind receptor-competent apo E polypeptides, and can therefore beuseful to determine receptor-competent apo E rather than total apo E.

Techniques for operatively linking an enzyme to an antibody molecule toform a conjugate are well known in the art. Exemplary techniques arediscussed in Maggio, Enzyme-Immunoassay, Chapter 4 by Kabakoff, CRCPress, Boca Raton, Fla. (1980), pages 71-104.

The monoclonal antibody molecules can be utilized as obtained fromhybridoma supernatants or as ascites. However, it is preferred thatpurified monoclonal antibody molecules be utilized.

Several means for purification of monoclonal antibody molecules are wellknown in the art and typically utilize chromatographic techniques. Fastprotein liquid chromatography (FPLC) is the purification technique ofchoice herein.

The enzyme-linked monoclonal antibody molecule conjugates are providedto the admixtures in the fluid phase. Those molecules are typicallydissolved in an aqueous composition. Typical compositions contain buffersalts as is the case of the exemplary purified monoclonalantibody-containing compositions used herein that includephosphate-buffered saline (PBS) as a diluent. Diluted ascites fluid alsois useful.

Preferably, non-specific protein binding sites on the surface of thesolid phase support are blocked. Thus, the solid phase-bound paratopicmolecules are bound as by adsorption or other well known means ofaffixation to the solid matrix. Thereafter, an aqueous solution of aprotein free from interference with the assay such as bovine, horse orother serum albumin that also is free from contamination with human apoB-100 or apo E is admixed with the solid phase to adsorb the admixedprotein onto the surface of the paratopic molecule-containing solidsupport at protein binding sites on the surface that are not occupied bythe monoclonal paratopic molecule.

A typical aqueous protein solution contains about 3 to about 10 weightpercent bovine serum albumin in PBS at a pH value of 7.1-7.5. Theaqueous protein solution-solid support admixture is typically maintainedfor a time period of at least one hour at 37 degrees C., and theresulting solid phase is thereafter rinsed free of unbound protein.

The vascular fluid sample can be plasma or serum, as already noted. Thesample is preferably diluted at about 1:500 to about 1:5000, and morepreferably at about 1:1000. The use of lesser dilution can provide toomuch of the apolipoprotein antigen to the admixture and impair thelinearity of the assay results as well as lower or abolish the solidphase-bound paratopic molecule excess over the admixed antigen. Use ofgreater than about a 1:20,000 dilution tends to decrease precision.

The maintenance times utilized can vary widely with little variance inresult so long as a minimum time of about 30 minutes at ambient roomtemperature (about 20-25 degrees C.) is utilized. Where it is desired touse a minimum 30-minute maintenance time, it is preferred that themaintained admixture be agitated during that time period to assuresubstantially complete immunoreaction between the apolipoprotein antigenand monoclonal paratopic molecules. Where longer maintenance times suchas one hour or more at room temperature are utilized, agitationtypically is not required. The desired agitation can be readily suppliedby means of a gyroshaker operated at about 100 rpm. Each of the assaysused in the method is capable of being carried out using immunoreactionadmixture maintenance times of about 30 minutes to about 60 minutes atambient room temperatures.

The amount of apolipoprotein antigen present in the assayedimmunoreactant is determined by admixture of the separated enzyme-linkedapolipoprotein-containing solid phase with a predetermined amount ofvisualizing reagent or reagents. Where HRPO is utilized as the enzymeindicating means, visualizing reagents such as hydrogen peroxide and anoxidative dye precursor such as o-phenylenediamine (OPD) present in anaqueous medium are admixed with the separated solid phase-boundimmunoreactant. The admixture so formed is maintained under biologicalassay conditions for a predetermined time such as at least about 30minutes at ambient temperature for color to develop. Color developmentis thereafter stopped by admixture of a stopping reagent such assulfuric acid. The optical density of the composition is thereafterread, compared to a standard curve value, and the amount ofapolipoprotein is determined, as is well known.

Thus, once the solid support and vascular fluid sample are prepared,each assay can be carried out at ambient room temperature in a timeperiod of about one hour; i.e., a 30-minute maintenance time withagitation for admixtures formed from both paratopic molecules and thesample aliquot, and another 30-minute maintenance time for colordevelopment. Indeed, one need not prepare the solid support just priorto each use, but rather, such supports as are described herein can beprepared and stored damp and covered under usual refrigerationconditions for a period of at least one month prior to use.

I. Diagnostic Systems

The present invention also contemplates a diagnostic system, typicallyin kit form, that can be utilized in carrying out the before-describedassay methods. The system includes, in an amount sufficient for at leastone assay, a subject apo E polypeptide and/or anti-apo E antibody ormonoclonal antibody of this invention as separately packagedimmunochemical reagents. Instructions for use of the packaged reagentare also typically included.

In one embodiment, a diagnostic system in kit form includes a solidsupport comprising a solid matrix such as a microtiter plate having ananti apo-E monoclonal antibody of this invention affixed thereto(operatively linked to the solid matrix) in an amount sufficient tocarry out at least one assay.

In preferred embodiments, the above diagnostic system further includes,as a separately packaged reagent, a second antibody, a reveal antibody,that contains antibody molecules that immunoreact with apo E-containinglipoprotein particles. In this embodiment, the reveal antibody canimmunoreact with any component present on the apo E-containinglipoprotein particle. Such particles include apo E/LDL, apo E/VLDL, apoE/HDL, and the like. Preferred are reveal antibodies that immunoreactwith apo B-100 because it is a conserved epitope on apo E-containinglipoprotein particles. Particularly useful anti B-100 antibody moleculesare the monoclonal antibodies secreted by the hybridomas MB47, which isavailable from the ATCC and has the accession number HB8746.

In another embodiment used for assaying a fluid sample for the presenceof apo E polypeptide or apo E-containing lipoprotein particles, adiagnostic system includes a solid support comprising a solid matrixhaving affixed thereto an apo E polypeptide of this invention. Thesystem can further include, as a separately packaged reagent, ananti-apo E polypeptide antibody of this invention for use in acompetition ELISA format.

Preferably, a diagnostic system further includes one or more of thefollowing: (i) a supply of hydrogen peroxide of known concentration;(ii) a visualizing oxidative dye precursor such as OPD; (iii) a solutionof a stopping agent such as 4 N sulfuric acid to quench thecolor-forming reaction; (iv) one or more buffers in dry or liquid formfor use in the assay; and (v) materials for preparing standard referencecurves.

As used herein, the term "package" refers to a solid matrix or materialsuch as glass, plastic, paper, foil and the like capable of holdingwithin fixed limits a polypeptide, polyclonal antibody or monoclonalantibody of the present invention. Thus, for example, a package can be aglass vial used to contain milligram quantities of a contemplatedpolypeptide or it can be a microtiter plate well to which microgramquantities of a contemplated polypeptide have been operatively affixed,i.e., linked so as to be capable of being immunologically bound by anantibody.

"Instructions for use" typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In preferred embodiments, a diagnostic system of the present inventionfurther includes a label or indicating means capable of signaling theformation of a complex containing a polypeptide or antibody molecule ofthe present invention.

The word "complex" as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms "label" and "indicating means" in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,"Immunofluorescence Analysis" in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I and ⁵¹ Cr representone class of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵ I. Another group of usefullabeling means are those elements such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ Nwhich themselves emit positrons. The positrons so emitted produce gammarays upon encounters with electrons present in the animal's body. Alsouseful is a beta emitter, such ¹¹¹ indium of ³ H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A "specific binding agent" is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA"format to detect the quantity of apo E in a vascular fluid sample suchas blood, serum, or plasma. "ELISA" refers to an enzyme-linkedimmunosorbent assay that employs an antibody or antigen bound to a solidphase and an enzyme-antigen or enzyme-antibody conjugate to detect andquantify the amount of an antigen present in a sample. A description ofthe ELISA technique is found in Chapter 22 of the 4th Edition of Basicand Clinical Immunology by D.P. Sites et al., published by Lange MedicalPublications of Los Altos, CA in 1982 and in U.S. Pat. Nos. 3,654,090;3,850,752; and No. 4,016,043, which are all incorporated herein byreference.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium although other modes of affixation applicable to proteinsand polypeptides well known to those skilled in the art, can be used.

Thus, in preferred embodiments, an apo E polypeptide, or anti-apo Eantibody molecule, of the present invention can be affixed in a solidmatrix to form a solid support that comprises a package in the subjectdiagnostic system.

Useful solid matrices are also well known in the art for preparing asolid support containing a reagent affixed thereto. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ);agarose; beads of polystyrene beads about 1 micron to about 5millimeters in diameter available from Abbott Laboratories of NorthChicago, IL; polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microtiter platesuch as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems. Suchmaterials include glass and plastic (e.g., polyethylene, polypropyleneand polycarbonate) bottles, vials, plastic and plastic-foil laminatedenvelopes and the like.

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

1. Polypeptide And Conjugate Preparation

a. Synthesis

The polypeptides p(141-155)₂, referred to as "tandem peptide",p(141-155)₃, the control polypeptides shown in Table 2, and otherdisclosed polypeptides were synthesized using the classical solid-phasetechnique described by Merrifield, Adv. Enzymol 32:221-96, (1969) asadapted for use with a model 430A automated peptide synthesizer (AppliedBiosystems, Foster City, California) using HOBt DCC activation.Polypeptide resins were cleaved by hydrogen fluoride, extracted andanalyzed for purity by high-performance liquid chromatography (HPLC)using a reverse-phase C18 column manufactured by Waters Associates,Milford, MA. The peptides were further purified by HPLC chromatographyon a Waters Auto500 preparative HPLC column. Amino acid compositionswere determined on a Beckman Model 6300 high performance analyzer andthe purity assessed by analytical HPLC. Peptide sequences, purity andamino acid composition of some of the polypeptides are shown in Table 6.Peptides were lyophilized and stored in a dark environment under vacuum.To prepare them for addition to the cultures, all peptides weredissolved in 10 mM Tris Hcl saline buffer and dialyzed overnight indialysis tubing with a molecular weight cut off of 1000, (SpectrumMedical, Los Angeles, CA).

                                      TABLE 2                                     __________________________________________________________________________    Designation.sup.1                                                                      Amino Acid Residue Sequence                                          __________________________________________________________________________    p93-112  LSKELQAAQARLGADMEDVR                                                 p139-149 SHLRKLRKRLL                                                          p141-150 LRKLRKRLLR                                                           p141-155 LRKLRKRLLRDADDL                                                      p145-154 RKRLLRDADD                                                           p150-160 RDADDLQKRLA                                                          p161-171 VYQAGAREGAE                                                          p167-176 REGAERGLSA                                                           p172-182 RGLSAIRERL                                                           p174-203 LSAIRERLGPLVEQGRVRAATVGSLAGQPL                                       p(LLRK).sub.4                                                                          LLRKLLRKLLRKLLRK                                                     p(LLRK).sub.8                                                                          LLRKLLRKLLRKLLRKLLRKLLRKLLRKLLRK                                     __________________________________________________________________________     .sup.1 The designation for each peptide indicates the position within the     amino acid residue sequence of the mature Apo E protein to which the          peptide sequence corresponds, i.e., is derived from.                     

The lysine residues of the dimer peptide were modified where indicatedby acetoacetylation with the diketene reagent as described by Weisgraberet al, J. Biol. Chem., 253:9053-9062, (1978). The arginine residues ofthe dimer peptide were modified by 1,2-cycloheximide modification asdescribed by Shive et al, Proc. Natl. Acad. Sci. USA, 83:9-13, (1986).

b. Self-Conjugation of Apo E Peptides p141-155 and p129-163

The synthetic peptides containing the amino acid residues 141-155 or129-163 of apo E or the peptide p(141-155)₂ were self-conjugated (i.e.,p141-155 was coupled to p141-155 and p129-163 was coupled to p129-163)according to the procedure of Hoare et al., J. Biol. Chem., 242:2447,(1967). Briefly, 100 mg of synthetic peptide was dissolved in 10 ml ofhigh purity water (Nanopure system). One gram of EDG[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride]wasadmixed to the peptide solution. The reaction proceeds rapidly at roomtemperature and is complete after one hour. During the first five to tenminutes the pH of the reaction admixture is monitored. The startingsolution, before the addition of EDG, is at a pH of approximately 3.7.Upon the addition of EDG, the pH increases to approximately 5.0 in thefirst five minutes. At five minutes, 50 ul of 0.1 N HCL is added. Nofurther addition of acid is necessary during the rest of the incubation.The admixture is rotated while incubating to insure complete reaction.The reaction is quenched with 10 ml of 2M acetate buffer, pH 4.75.

To isolate conjugated (operatively linked) peptides from unreactedpeptide and chemicals, the self-conjugation preparation is dialyzed in2,000 molecular weight cut-off dialysis tubing, 18 millimeter wide andapproximately two times the length of the sample volume. Startingdialysis is done against 2M acetic acid. A gradient reducing theconcentration of acetic acid from 2M to <0.01M is achieved by changingthe dialysis buffer (one liter) every hour stepping down theconcentration of acetic acid by 50% at each change. Once the acetic acidconcentration has been lowered to <10mM, the sample is then dialyzedagainst one liter highly purified water over night at room temperaturewith at least five one liter changes. The sample is lyophilized todryness and redissolved in phosphate-buffered saline (PBS) and is readyfor addition to cell culture.

The yield of self-conjugated peptide from this procedure is low, usuallynot greater than 5%. For instance, in one conjugation starting with 100mg of synthetic peptide, 2.82 mg of p141-155 self-conjugated peptide wasrecovered. The polypeptide concentration of the peptide redissolved inPBS was determined by the Lowry protein assay method. All additions ofpeptide to cell culture of this self-conjugated preparation were basedon the value from Lowry assay. The activity in the self-conjugatedp141-155 preparation is stable for at least two months when stored at-20C.

2. Monomeric Peptides And Peptide-BSA Conjugates Do Not SpecificallyInhibit Lymphocyte Proliferation

A lymphocyte cell culture system was used to examine the ability ofvarious polypeptides and conjugates to mimic the ability of Apo E toinhibit lymphocyte differentiation as evidence by proliferation.

Human peripheral blood mononuclear (PBM) cells are isolated from wholeblood on a Ficoll-Hypaque gradient. The collected PBMs are washed threetimes with fresh medium (RPMI-1640 plus 5% fetal bovine serum,glutamine, penicillin, streptomycin and HEPES buffer). After washing,the cells are counted and set at a density of 1×10⁵ /ml. For culture,0.2 ml of the cells are plated per well of a 96-well microtiter plate.The peptide or conjugate is added to the wells in a 50 ul volume thatcan be either filter or UV sterilized. The typical timing for thisexperiment is to expose the cells to the Apo E polypeptide orself-conjugate over night. PHA, the mitogen that stimulates lymphocytegrowth and proliferation, is added the next day. Forty-eight hours afterthe addition of PHA, 1 uCi of ³ H-thymidine is added per well in a 1 ulvolume and 18-24 hours later the cells are harvested on a mash. The mashcell harvester functions to collect the cells on filter paper where theyare washed with buffer to remove free ³ H-thymidine. The filter papersare dried, put into scintillation vials with scintillation cocktail andthe B emissions obtained. Each data point collected represents theaverage thymidine uptake in four wells, and the standard error was lessthan 10% for all data points.

When examined in the above-described assay, the polypeptides shown inTable 2 had no effect on lymphocyte proliferation when used innon-conjugated, monomeric form. However, when those same polypeptideswere used conjugated to bovine serum albumin (BSA), lymphocyteproliferation was inhibited in an equivalent manner by all peptidesstudies, as evidenced by decreasing amounts of thymidine uptake withincreasing dose of conjugate. A typically linear inhibition was observedfrom about 50 to 75×10³ counts per minute (cpm) at about 6 micrograms(μg) per milliliter (ml) peptide-BSA to about 5 to 40×10³ cpm at about200 μg/ml.

3. Multimeric Peptides And Peptide Self-Conjuqate's Specifically InhibitLymphocyte Proliferation In A Non-Cytotoxic Manner

The ability of polypeptides and conjugates of this invention tospecifically inhibit lymphocyte proliferation was examined using theassay described in Example 2. Specifically, self-conjugates(p172-182)-(p172-182), (p93-112)-(p93-112) and (p141-155)-(p141-155)were compared for the ability to inhibit lymphocyte proliferation. Inthis assay, self-conjugates (p172-182)-(p172-182) and(p93-112)-(p93-112) demonstrated no significant capacity to inhibitlymphocyte proliferation, as evidenced by their failure to reduce H³-thymidine uptake. In contrast, a self-conjugate of this invention,(p141-155)-(p141-155) began demonstrating significant (detectable)inhibitory activity in the concentration range of about 0.8 to about 1.0μg/ml, and exhibited complete inhibition (less than 2% of control) abovea concentration of about 2.5 μg/ml.

To examine whether or not the observed inhibition of proliferation wasdue to cell death (direct cytotoxicity), the treated cell cultures weresubjected to a lactate dehydrogenase (LDH) release assay as described byCarney et al., J. Immunol., 134:1084, (1985). The presence of LDH in thecell culture supernatant indicates toxicity because it is released bycells that have lysed.

1×10⁶ peripheral blood mononuclear cells per ml were cultured at 37C inRPMI with 5% fetal bovine serum in the presence of self-conjugate(p141-155)-(p141-155). All cells were then exposed to PHA at 24 hr, werelabeled with ³ H-thymidine at 48 hr and were harvested at 72 hr. Cellculture supernatants from duplicate wells were collected for assay oflactate dehydrogenase (LDH) activity according to the method of Carneyet al., J. Immunol., 134:1804, (1985). Data is expressed as percent ofcontrol. ³ H-thymidine uptake and LDH activity of PBS-exposed controllymphocytes was 0,085 and 1.232 change in O.D. 340 nm/min, respectively.LDH activity was measured following lysis of the cells with H₂ O. Eachpoint measured represents the average value from 4 wells per treatmentand the standard error was less than 10%.

According to the results of this study, for self-conjugate(p141-155)-(p141-155) there is less than 4% release of LDH activity atthe concentration of self-conjugate that inhibits lymphocyteproliferation by greater than 95% (4 μg/ml). These results demonstratethat the polypeptides and self-conjugates of this invention are notdirectly cytotoxic and inhibit lymphocyte proliferation specifically.

Inhibition by Apo E of mitogen-stimulated lymphocyte proliferation isnot readily reversible. The reversibility of the inhibition by theself-conjugated peptides of this invention was tested by incubatinglymphocytes overnight with the peptides, then washing the culturesbefore adding PHA. As shown in Table 3 the self-conjugate (p141-155) -(p141-155), was maximally inhibitory under these conditions. Further,this inhibitory activity could not be reversed by washing away thenon-cell-associated self-conjugate peptide prior to PHA stimulation. Inthis respect, the inhibitory activity of self-conjugate (p141-155)-(p141-155) mimicked the irreversible inhibitory activity observed withnative Apo E.

                  TABLE 3                                                         ______________________________________                                        Lymphocyte Proliferation was Irreversibly Inhibited                           by a Self-Conjugate of Peptide 141-155.sup.a                                              3H-Thymidine Uptake (cpm ± SD)                                 Treatment     No Washing  With Washing.sup.b                                  ______________________________________                                        PBS           69,106 ± 6,555                                                                         57,123 ± 8,842                                   Self-conjugated                                                                             1,035 ± 170                                                                             425 ± 100                                       apo E.sub.141-155.sup.c                                                       Self-conjugated                                                                             83,480 ± 4,027                                                                         64,524 ± 4,200                                   apo AI.sub.95-105.sup.c                                                       ______________________________________                                         .sup.a All cultures contained 1 × 10.sup.6 PBM cells per ml. The        cells were cultured at 37° C. in RPMI 1640 with 5% fetal bovine        serum. All cells were exposed to PHA at 24 hr., labeled with .sup.3           Hthymidine at 48 hr. and harvested at 72 hr.                                  .sup.b At 24 hr. after conjugates were added, prior to PHA stimulation,       the cells were washed three times in medium and resuspended to their          original volume in RPMI 1640 containing 5% serum.                             .sup.c Peptides were used at a final concentration of 40 μg/ml.       

4. Monomeric Peptides And Peptide-BSA Conjugates Do Not SpecificallyInhibit Ovarian Androgen Production

The cell system used to examine the effect of various peptides andconjugates on ovarian androgen production was that described by Curtisset al., J. Biol. Chem., 263:10965, (1988). Briefly, hypophysectomized(pituitary removed) immature female rats are sacrificed by cervicaldislocation. The ovaries taken from the rats were trimmed free of fatand other non-ovarian tissues, cut into approximately six to eightpieces per ovary and dissociated in a solution of collagenase Dnaasewhich degrades the connective tissue releasing cell clusters.Approximately one to two hours of 37C incubation is required todissociate the tissue. The cells were washed three times with freshMcCoy's 5A modified medium supplemented with glutamine andpenicillin/streptomycin. No serum was added to this medium. The cellswere cultured over night in 10 ml of medium in a T-25 flask to allow thenon-steroidogenic cells to adhere. The next day non-adherent cells wererecovered, washed three times with fresh media, counted and plated at adensity of 1×10^(3/) ml aliquotting 0.2 ml per well of a 96-wellmicrotiter plate. All media in the experiment contains luteinizinghormone (LH) at 4 ng/ml and human high density lipoprotein (HDL) at 300μg protein/mi. Monomeric peptides, peptide-BSA conjugates andself-conjugated peptide preparations at various concentrations (0.2 to50 μg/ml) were added to the cells. The cells were then cultured for 48hours at 37C, the supernatants were recovered, transferred to a clean96-well microtiter plate and kept at -20C until the concentrations ofandrostenedione and progesterone were measured by radioimmunoassay. Eachdata point represents the average of measured androstenedioneconcentrations in four wells per treatment, and the standard error wasless than 10% for all data points.

When examined in the above-described assay, the polypeptides p139-149,p141-155, p145-154, p150-160, p161-171, p167-176, p172-182 and p174-203,having sequences shown in Table 2 were studied, and when used innon-conjugated, monomeric form, had no effect on androgen secretion.However, when those same peptides were used in the form of a conjugateto BSA, androgen production was inhibited in an approximately equivalentmanner by all peptides studied thereby indicating a lack of specificity,as evidenced by decreasing amounts of androstenedione production withincreasing dose of conjugate. Typically linear and decreasing productionwas observed from about 14 to 28 nanograms (ng) androstenedione per mlat about 0.2 μg BSA-peptide per ml to about 4 to 13 ng androstenedioneper ml at about 15 μg BSA-peptide per ml. PBS control exhibited 22.1 ngandrostenedione per ml.

5. A Multimeric Peptide And A Peptide Self-Conjugate SpecificallyInhibit Ovarian Androgen Production

The ability of polypeptides and conjugates of this invention tospecifically inhibit ovarian androgen production was examined using theassay described in Example 4. In this assay, self-conjugate(p141-155)-(p141-155) demonstrated the ability to inhibit androgenproduction in ovarian theca/interstitial cells at a concentration as lowas about 1.5 μg/ml, and exhibited less than 20% of control production at10 μg/ml. In contrast, self-conjugates p(93-112)-p(93-112) orp(172-182)-p(172-182) not containing the Apo E 141-155 do not inhibitandrogen production in the above concentrations.

Apo E inhibition of ovarian cell androstenedione production isreversible. To test if the inhibitory activity of self-conjugate(P141-155) -(P141-155) was reversible, ovarian cells were cultured withthe peptide for 48 hours. At the end of this culture, the medium wasremoved and replaced with fresh medium without the self-conjugatedpeptide. As shown in Table 4, ovarian androstenedione production wasinhibited during the first 48 hours of culture but, after the peptidewas removed and the ovarian cells refed with fresh medium, theirandrostenedione production during the subsequent 48 hours returned tocontrol levels. In this respect, the activity of the polypeptides and/orconjugates of the present invention mimicked the activity of the nativeAPO E.

                  TABLE 4                                                         ______________________________________                                        Ovarian Androgen Production was Reversibly Inhibited                          by a Self-Conjugate of Apo E peptide 141-155.sup.a                                        Androstenedione Accumulation                                                  (ng/ml)                                                                         First 48 hr.                                                                              Second 48 hr.                                       Treatment     of Culture  of Culture                                          ______________________________________                                        PBS           12.13 ± 0.26                                                                           11.27 ± 0.84                                     Self-conjugated                                                                              6.27 ± 1.26                                                                           13.34 ± 2.75                                     Peptide                                                                       ______________________________________                                         .sup.a Ovarian theca/interstitial cells (20,000/0.25 ml) were cultured in     the absence or presence of selfconjugated peptide. After 48 hr. of cultur     the supernatants were removed and the cells refed with fresh medium           without selfconjugated peptide.                                          

To examine whether or not the observed inhibition of androgen productionwas due to cell death (cytotoxicity), the treated cell cultures wereassayed for progesterone production as described by Dyer et al., J.Biol. Chem., 263:10965-10973, (1988). The presence of progesterone inthe culture indicates the cell are viable. Briefly, increasingconcentrations of self-conjugate (p141-155)-(p141-155) were added toovarian cells (1×10⁵ /ml) that were cultured at 37C in serum-freeMcCoy's 5a medium containing 4 ng/ml of LH and 300 μg/ml of human HDL.After 48 hr of culture the supernatants were collected, androstenedioneand progesterone (steroid) concentrations measured by radioimmunoassay,and the data collected exhibited a standard error of less than 10% forall points studied when measured in quadruplicate. According to theresults of this study, for self-conjugate (p141-155)-(p141-155) therewas no significant decrease in progesterone production at theconcentrations of self-conjugate (5-10 μg/ml) that inhibit 95% ofandrostenedione (Adione) production. This result indicates that theobserved inhibitory activity was specific and not the result of celldeath (cytotoxicity).

6. A Dimeric Peptide and Self-Conjugates of the Dimeric Peptide AffectLymphocyte Proliferation

The ability of the dimeric peptide p(141-155)₂ and self-conjugates ofthis peptide p(141-155)₂ - p(141-155)₂ to affect lymphocyteproliferation was examined using the assay described in Example 2.

Various concentrations of various Apo E peptide were added to culturesthat contained 8×10⁵ peripheral blood mononuclear cells per ml. Thecells were cultured at 37C and RPMI with 5% serum. All cells wereexposed to PHA at 24 hours, labeled with 3H-thymidine at 72 hours andharvested at 90 hours. The counts per minute (cpm) of 3H-thymidine inthe harvested cells was measured, and the data are expressed as the meanof four wells per treatment as a measure of thymidine uptake (x 1000cpm). The standard deviation was less than 10% for all means.

Tandem Apo E peptide p(141-155)₂ enhanced lymphocyte proliferation atlow concentrations and inhibited lymphocyte proliferation at highconcentrations. This ability to both enhance and inhibit lymphocyteproliferation, depending upon the dose of the peptide, mimics theactivity of the native Apo E and exhibits a dose-dependent biphasicmanner of affecting lymphocyte proliferation. Enhancement of lymphocyteproliferation was seen at doses of tandem Apo E peptide from about 1μg/ml to about 3 μg/ml, while inhibition of proliferation was seen atconcentrations of at least about 3 μg/ml and up to about 30 μg/ml.Self-conjugates of tandem Apo E peptide p(141-155) 2p(141-155)2 wereeven more potent than tandem Apo E peptide alone in its ability toaffect lymphocyte proliferation with enhancement occurring as low asfrom about 0.2 to 1.0 μg/ml and inhibition occurring over the range ofabout 1.5 to 4 μg/ml.

7. A Dimeric Peptide and Self-Conjugates of this Peptide Affect OvarianAndrogen Production

The ability of tandem Apo E peptide p(141-155)2 and self-conjugates ofthis peptide to affect ovarian androgen production was examined usingthe assay described in Example 4. Increasing concentrations of tandemApo E peptide were added to ovarian cells (8×10⁴ /ml) that were culturedat 37C in serum-free McCoy's 5a modified medium containing 4 ng/ml of LHand 300 μ/ml of human HDL. After 48 hours of culture, the supernatantswere collected, the androstenedione concentration measured byradioimmunoassay. When tandem Apo E peptide was added in concentrationsless than about 0.04 μg/ml, androstenedione production was enhanced.Concentrations of tandem Apo E peptide greater than about 0.2 to 2 μg/mlinhibited androstenedione production in a dose-dependent manner.

8. Apo E polypeptide binds Lipoproteins

To assess the degree of binding of the tandem peptide with lipoproteins,¹²⁵ I-tandem peptide was mixed with very low density lipoprotein (VLDL),LDL, high density lipoprotein (HDL) or lipoprotein-depleted serum (LPDS)to measure direct binding. Briefly, the lipoproteins were isolated byultracentrifugation according to Curtiss, et al., J. Biol. Chem.,257:15213 (1982). The lipoprotein-depleted serum (LPDS) was obtained asthe bottom fraction remaining after flotation of the HDL at a density of1.25 gm/ml. All fractions were diluted in PBS containing 3% bovine serumalbumin (BSA). The tandem peptide p(141-155)₂ was radiolabeled using theiodine monochloride method of Brown et al., Methods Enzymol., 98:241(1983) to a specific activity of 7.99×10⁶ cpm/μg and was 99%precipitable in 10% trichloroacetic acid (TCA). The binding assays wereperformed in siliconized tubes in a total volume of 0.3 ml of PBS thatcontained 3% BSA, 20 μg of lipoprotein or LPDS and 250 ng of ¹²⁵I-tandem peptide. After 1 hr at 37° C. the lipoprotein bound peptide wasseparated from the free peptide. Tubes containing VLDL, LDL and LPDSwere precipitated with 0.3 ml of 555 uM phosphotungstic acid and 25 mMMgCl₂ and tubes containing HDL was precipitated with 0.3 ml of an apoAI-specific antiserum. Each precipitation condition had been previouslyoptimized with the use of radioiodinated lipoproteins and was designedto achieve 100% precipitation of the lipoproteins. Precipitatedradioactivity was measured by detecting gamma radiation and expressedbased on specific activity as nanograms (ng) of peptide bound permicrogram (μg) of protein.

The results, shown in Table 5, indicate that less than 0.7% of the added¹²⁵ I-tandem peptide was associated with HDL, and that 58% and 39% ofthe added peptide was found associated with VLDL and LDL, respectively.Calculated as the number of tandem peptide molecules bound perlipoprotein particle, VLDL, LDL and HDL each contained 1.8, 0.25 and0.0043 peptide molecules per particle, respectively. Therefore, the apoE polypeptide p(141-155)₂ preferentially binds lipoprotein particles inthe following order VLDL >LDL >HDL.

                  TABLE 5                                                         ______________________________________                                                      ng of .sup.125 I-tandem                                                                    Molar ratio of                                     LIPOPROTEIN   peptide bound                                                                              peptide per lipo-                                  OR PROTEIN    per μg protein                                                                          protein particle                                   ______________________________________                                        VLDL          7.25         1.8                                                LDL           1.87         0.25                                               HDL           0.07         0.0043                                             LPDS          0.76         --                                                 ______________________________________                                    

9. A Tandem Peptide Affects LDL Binding and Degradation

The LDL receptor binding properties of the tandem peptide were assessedusing normal human fibroblasts and a transformed human monocytic-likecell line, THP-1 as a source of LDL receptor. THP-1 cells were studiedbecause both the LDL receptor and another lipoprotein receptor, thescavenger receptor, are present and binding with those receptors iseasily examined. In the undifferentiated state THP-1 cells express LDLreceptors that are regulated by the lipoprotein content of the culturemedium; Hara, et al., Biochem. Biophys. Res. Commun., 146:802 (1987);Via, et al., J. Lipid Res., 30:1515 (1989). Following phorbol myristateacetate ester (PMA)-stimulation the cells stop dividing, differentiateinto macrophage-like cells, eventually lose most of their LDL receptorsand acquire scavenger receptors that bind acetylated or modified LDL;Hara, et al., Biochem. Biophys. Res. Commun., 146:802 (1987); Via, etal., J. Lipid Res., 30:1515 (1989).

a. Inhibition of degradation by unstimulated THP-1 cells

THP-1 cells were acquired from American Type Culture Collection andcultured in serum-free RPMI-1640 medium supplemented with 1%Nutridoma-HU for 48 hr to upregulate the LDL receptors. LDL was isolatedby ultracentrifugation and radiolabeled using the iodine monochloridemethod as described in Example 8 to a specific activity 200-300 cpm/ng.Greater than 99% of the ¹²⁵ I-LDL ligand was precipitable by 10% TCA.The peptides indicated in FIGS. 1A and 1B were synthesized, as describedin Example 1, purified to >95% by high pressure liquid chromatographyand the amino acid composition verified. All peptides were solubilizedin PBS. "AI 74-105"is a control peptide derived from residues 74-105 ofapolipoprotein AI, "Monomer" is p(141-155), "Tandem Peptide" isp(141-155)₂, "LDL" is unlabelled LDL, and "aLDL" is acetylated LDL.

Binding and degradation of LDL was evaluated as the disappearance ofacid soluble ¹²⁵ I-LDL radioactivity from the incubation over a 5-hourincubation at 37C. Various concentrations of unlabeled LDL or tandem apoE peptide were coincubated with the ¹²⁵ I-LDL to determine their effectson binding and degradation.

For assay, 5×10⁵ THP-1 cells in 0.5ml DMEM were co-incubated in a 1.5mlmicrofuge tube with 2-5 μg/ml of ¹²⁵ I-LDL at 37° C. in the presence ofvarious concentrations of competitors indicated in FIGS. 1A and 1B.After 5 hr the cells were pelleted at 5000 x g, the supernatants wereremoved and the supernatants were extracted with 10% TCA and counted toform a TCA soluble cpm. The results were expressed as B/Bo where Bequals the total TCA soluble cpm in supernatant collected in thepresence of LDL or peptide and B_(o) equals the total TCA soluble cpm insupernatant of phosphate buffered saline (PBS) treated control cells.Each point is the average of 4 replicates with standard error of themean (SEM),10%.

As shown in FIG. 1A, the tandem apo E peptide p(141-155)₂ enhanced LDLdegradation by unstimulated THP-1 cells at low concentrations andinhibited LDL degradation at high concentrations, i.e., the effect wasbiphasic. In this experiment, enhancement of LDL concentration occurredat tandem apo E peptide concentrations ranging from about 0.08 to about1.5 μM. Inhibition of LDL degradation began to occur at tandem apo Epeptide levels of 2.0 to 5.0 μM. Unlabeled LDL inhibited ¹²⁵ I-LDLdegradation, indicating the specificity of the LDL receptor.

It can be seen that neither a control apo A-I 74-105 peptide or themonomer apo E 141-155 peptide LDL inhibited ¹²⁵ I-LDL degradation. Incontrast, the tandem apo E peptide inhibited ¹²⁵ I-LDL degradation by50% at 5uM. Approximately a 200-fold molar excess of the tandem apo Epeptide compared with LDL was required to achieve 50% inhibition ofdegradation.

b. Inhibition is LDL receptor-specific

To verify that the inhibition of LDL degradation was specific for theLDL receptor, the effect of the tandem peptide on scavenger receptorprocessing of acetylated LDL (aLDL) was tested.

THP-1 cells (5×10⁵ in 1.0ml of DMEM medium per 16 mm culture dish) werestimulated with PMA (10⁻⁷ M) for 4 days. LDL was acetylated (Hara, H. etal., Biochem. Biophys. Res. Comm., 146(2):802-808, 1987) and thenradio-labeled as described in Example 8 to a specific activity of300-500 cpm/ng resulting in ¹²⁵ I-aLDL that was greater than 99%precipitable by 10% TCA. The cells were washed with PBS and the assayperformed in DMEM medium supplemented with 1% Nutridoma-HU plus 25 μg/mlof ¹²⁵ I-aLDL. After 5 hr at 37° C., the supernatants were removed fromthe wells and extracted with 10% TCA. The B/B₀ ratios were analyzed asdescribed above. The results are depicted in FIG. 1B.

Neither LDL nor the tandem peptide inhibited the degradation of ¹²⁵I-aLDL by PMA-stimulated THP-1 cells. As can be seen in FIG. 1B, at 12.5uM the tandem apo E peptide caused an 80% inhibition of ¹²⁵ I-LDLdegradation but had no effect on ¹²⁵ I-aLDL degradation.

10. Confirmation of binding by the LDL receptor and Identification ofcritical amino acids

The role of specific amino acids within residues 141-155 of intact apo Ehas been studied by Lalazar et al., J. Biol. Chem., 263:3542 (1988) withhuman apo E variants containing single amino acid substitutions. Thevariants were produced in a bacterial expression system complexed withthe phospholipid, dimyristoylphosphatidylcholine (DMPC) and tested fortheir ability to bind to fibroblast LDL receptors. Lalazar et al., J.Biol. Chem., 263:3542 (1988). To confirm that the tandem peptide wasbound by the LDL receptor and to identify amino acids within thispeptide that were critical for binding, three tandem peptides wereprepared as described in Example 1 that contained the same amino acidsubstitutions at both positions in the tandem sequence. The changesincluded substitutions of the basic amino acids Lys 143 to Ala, Arg 150to Ala, and Leu 144 with an alpha helix disrupting amino acid, Pro. ¹²⁵I-LDL at a specific activity of 700-900 cpm/ng (>99% precipitable by 10%TCA) was used for the binding assay as described in Example 9. Normalfibroblasts were plated in 16 mm culture dishes, grown for 72-96 hr inDMEM medium with 10% fetal bovine serum (FBS) and then transferred toDMEM medium with 10% lipoprotein-depleted serum (LPDS) for 48 hr toupregulate the LDL receptors. The amount of ¹²⁵ I-LDL bound per well wasnormalized to the protein content of each well, which was measured withthe Bio-Rad protein assay reagent using bovine serum albumin (BSA) as astandard.

As illustrated in FIG. 2, each of the substitutions in p(141-155)₂reduced the ability of the tandem peptide to inhibit the binding of ¹²⁵I-LDL to fibroblasts. The Lys 143 to Ala substitution had the smallesteffect, whereas the Leu 144 to Pro substitution had the greatest impact.In fact, the Leu 144 to Pro substituted tandem peptide had no activityat 60 μM, an effect that was similar to that observed with the nativeapo E variant. Lalazar et al., J. Biol. Chem., 263:3542 (1988). Thusmultimers of the apo E peptides that define an LDL receptor bindingmoiety can have substitutions of Lys (143) to Ala or Arg (150) to Alawithout significant alterations in the biological activity of themultimers.

11. Higher ordered multimers of the 141-155 sequence may have greaterbinding activity

A trimer of the p141-155 peptide, p(141-155)₃, was synthesized as inExample 1 and its activity compared with p(141-155) (monomeric) and withp(141-155)₂ (dimeric) peptide. Normal fibroblasts were plated in wellsof a 96-well culture plate and grown for 96 hr in DMEM medium with 10%FBS. The cells were transferred to DMEM medium with 10% LPDS for 48 hrto up regulate the LDL receptors. The LDL degradation assay wasperformed according to Example 9 with 2 μg/ml of ¹²⁵ I-LDL (300-500cpm/ng) in 0.2 ml/well in DMEM medium with 10% LPDS and incubated for 5hr at 37° C. The supernatants removed from the wells were extracted with10% TCA. The TCA soluble counts were normalized to protein content perwell as measured by the Bio-Rad protein assay.

Compared on a molar basis for their ability to inhibit fibroblast ¹²⁵I-LDL degradation, the trimer peptide was 15 times more effective thanthe dimeric peptide (FIG. 3). The results indicate that higher orderedmultimers of the 141-155 sequence have increased receptor bindingactivity.

12. Total number of binding sites per cell

The direct binding of the ¹²⁵ I-tandem peptide [p(141-155)₂ ] todividing THP-1 cells was measured. THP-1 cells were cultured for 48hours in LDL-free RPMI-1640 medium containing 1% Nutridoma-Hu.Increasing amounts of ¹²⁵ I-tandem peptide (17,444 cpm/pmole), labeledas described in Example 8 were added to 5×10⁵ cells/0.05 ml of DMEMmedium containing 1% Nutridoma-Hu combined with 0.05 ml of PBS or 0.05ml of VLDL 500 μg/ml in siliconized glass tubes. After 20 min at 4° C.the free ¹²⁵ I-tandem peptide was separated from the bound peptide bysedimenting the cells through silicone oil. Background counts were <1000cpm/tube.

¹²⁵ I-tandem peptide binding was found to be linear with cell number,saturable and reached an apparent steady state within 20 min at 4° C.¹²⁵ I-tandem peptide binding to THP-1 cells was specifically inhibitedby apo B- and apo E-containing lipoproteins with the ability to inhibitordered as follows:

VLDL>LDL>HDL.

The binding of the ¹²⁵ I-tandem peptide to the cells also was dependenton Ca⁺⁺. A 2-fold increase in the amount of ¹²⁵ I-tandem peptide boundto the cells was obtained when the Ca⁺⁺ concentrations were increasedfrom 0.1 to 2.0 mM, whereas Mg⁺⁺ at up to 2.0 mM had no effect. Finally,if THP-1 cells were deprived of LDL-containing serum for 96 hr before¹²⁵ I-tandem peptide binding was assayed, a 2-3 fold increase wasobserved with both ligands, indicating up-regulation of both the LDLreceptor and the tandem peptide binding site.

When the binding of the ¹²⁵ I-tandem peptide to THP-1 cells cultured inLDL-free medium was studied in the presence of 2.0 mM Ca⁺⁺ and in theabsence and presence of 500 μg/ml of VLDL, specific saturable bindingwas observed with a Kd of 1.1×10⁻⁷ M. Nonspecific binding (i.e., bindingin the presence of 500 μg/ml VLDL) averaged 23.4% of the total countsbound. From this data the total number of binding sites per cell wascalculated to be 1500.

Although the data indicates that the tandem peptide bound the LDLreceptor, the LDL receptor may not be the only fibroblast or THP-1binding site for the tandem peptide. Low density lipoproteinreceptor-related protein (LRP) is a recently described cell surfaceprotein that contains reiterated sequences, which are homologous tosimilar sequences in the LDL receptor; Herz et al., EMBO J., 7:4119(1988); Beisiegel, et al., Nature, 341:162 (1989). Recent studiesindicate that LRP may only interact with apo E-enriched lipoproteinsincluding beta-VLDL; Kowal, et al., Proc. Natl. Acad. Sci. USA, 86:5810(1989). Unlike the LDL receptor, the LRP receptor on fibroblasts doesnot appear to be down regulated by exposure of the cells to LDL; Kowal,et al., Proc. Natl. Acad. Sci. USA, 86:5810 (1989).

13. Therapeutic Application

Specific targeting of an apo E polypeptide such as the tandem peptide tocholesterol-rich lipoproteins is accomplished by designing a syntheticpeptide that has high affinity lipid and receptor binding properties.Attachment of the 141-155 tandem sequence p(141-155)₂ to a lipophilicmolecule or peptide facilitates a specific high affinity association ofthe tandem sequence with cholesterol-rich lipoproteins and increases thehepatic clearance of the lipoprotein from the circulation. Areproducible and significant enhancement of LDL degradation by THP-1cells (FIG. 1A) and by fibroblasts (FIG. 3) at low concentrations of thetandem peptide was observed when tandem peptide was contacted with thecells in vitro.

There are several examples cited in the literature in which it has beenobserved that apo E function is dependent on the number of apo Emolecules per lipoprotein particle: (a) reducing the number of apo Emolecules per DMPC complex results in decreased binding; Mahley, et al.,Biochim. Biophys. Acta, 737:197 (1983); (b) VLDL uptake by the liver isenhanced by adding additional copies of thrombin-accessible apo E;Bradley, et al., J. Biol. Chem., 259:14728 (1984); (c) an average ofgreater than one apo E molecule per particle is required for VLDL tostimulate macrophage cholesterol esterification; Soltys, et al., J.Lipid Res., 29:191 (1988); (d) VLDL carrying more apo E are removed fromblood more rapidly; Yamada, et al., Proc. Natl. Acad. Sci. USA, 86:665(1989), and finally (e) only apo E-enriched beta VLDL is taken up by theLRP receptor; Kowal, et al., Proc. Natl. Acad. Sci. USA, 86:5810 (1989).It is believed herein that this requirement reflects the closeassociation of at least two copies of apo E to form an LDLreceptor-competent apo E ligand. The discoveries disclosed hereinestablish that various numbers of copies of the apo E can be utilized.

14. Preparation of Polyclonal Antisera to Synthetic Polypeptides

a. Preparation of Immunogen

LDL was isolated from plasma obtained by plasmaphoresis of normal pooledrabbit blood (Scripps Clinic and Research Foundation Vivarium, La Jolla,Calif.). Plasma so obtained was adjusted to contain a finalconcentration of 2 millimolar (mM) benzamidine, 14 mMethylenediaminetetraacetic acid (disodium salt) (EDTA), 20 microgramsper milliliter (μg/ml) soybean trypsin inhibitor, 10,000 units per mlaprotinin, 20 μg/ml lima bean trypsin inhibitor, 25 μg/ml polybrene, and1 μM D-phenylalanyl-1-prolyl-1-arginine chloromethyl ketone (PPACK). TheLDL was then isolated from this adjusted plasma by sequentialultracentrifugation using solid potassium bromide (KBr) for densityadjustment.

First, the adjusted plasma was centrifuged at 186,000×g for 18 to 24hours at 4 degrees centigrade (4° C.). The top layer of the resultingsupernatant containing apo/VLDL was removed and retained. The bottomlayer of the supernatant was recovered and admixed with solid KBr layeruntil the density was greater than 1.063 grams per milliliter (g/ml).The resulting admixture was then layered under a 0.1% EDTA solutioncontaining KBr at density of 1.063 g/ml and centrifuged at 186,000×g for18 to 24 hours.

After the second centrifugation, the top layer containing LDL wasrecovered and the bottom layer containing HDL was discarded. The toplayer is admixed with solid KBr until the density was adjusted togreater than 1.063 g/ml. That adjusted layer was layered under a 0.1%EDTA solution containing KBr at a density of 1.21 g/ml and wascentrifuged at 186,000×g for 18 to 24 hours at 4° C.

The top layer was then recovered, and solid KBr was admixed until thedensity was greater than 1,063 g/ml. That adjusted top layer was layeredunder a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml,and still further centrifuged at 250,000 ×g for 18 to 24 hours at 4° C.The top layer containing concentrated LDL was recovered and dialyzedagainst PBS (phosphate-buffered saline, pH 7.2) and stored at -70° C.

The multimeric polypeptide analogs of apo E, (p141-155)₂, (p141-155)₃,and self-conjugates of these, were synthesized as described in Example1a and b. The apo E polypeptide p(141-155)₂ was dissolved in 1.5 Msodium acetate, pH 7.8, to a final concentration of 6 mg/ml in a totalvolume of 5 mls. A dissolved polypeptide was admixed with 2.5 ml each ofa 2 mg/ml LDL solution and a 3 M sodium acetate solution, pH 7.8, for apeptide:LDL ratio of 1000:1. Added to the polypeptide and LDL reactionmixture was a 500 mM glutaraldehyde solution using a 2.7 molar excess ofglutaraldehyde to peptide. The admixture was maintained at roomtemperature for 10 minutes, after which a 40 mM solution of sodiumborohydride was added to a final concentration of 0.2 mM. The admixturewas thereafter maintained at 37° C. for 5 to 8 hours, followed bydialysis against PBS for 5 days with two buffer changes per day, usingdialysis tubing having a 12,000 to 14,000 molecular weight cut-off. Thedialysed solution was centrifuged at 2500×g for 10 minutes, and theresulting pellet was resuspended in 5 ml PBS to form a peptide-LDLimmunogen. A peptide-LDL immunogen was prepared using theabove-described polypeptide, namely p(141-155)₂.

b. Immunization and Collection of Polyclonal Antisera

The peptide-LDL immunogen prepared in Example 14a above was emulsifiedusing the Ribi Adjuvant System (Ribi Immunochem Research, Inc.,Hamilton, Mont.) according to the manufacturer's instructions. Thepeptide-LDL antigens were incorporated into the emulsion at aconcentration of 300 μg/ml. After pre-immune serum samples werecollected, two rabbits were injected with 1 ml of a prepared emulsion.The 1 ml emulsion dose was administered as follows: 0.30 mlintradermally (0.05 ml in each of 6 sites); 0.40 ml intramuscularly (0.2ml into each hind leg); 0.10 ml subcutaneously (in the neck region); and0.20 ml intraperitoneally. The rabbits were injected 6 times atthree-week intervals following the injection protocol as detailed. Atone week after the second through sixth injections, blood samples werecollected to check antibody titer against the specific peptide used asthe immunogen by the SPRIA assay described below. The collected bloodsamples were stirred in a 37° C. oven for 1 hour, after which thesamples are centrifuged at 3000×g for 20 minutes. The interface wascollected and spun in a microfuge at 12,000×g for 5 minutes. Thesupernatant containing anti-peptide antibodies was collected and storedat -20° C.

The peptide antibody titers were determined by solid phaseradioimmunoassay (SPRIA) essentially as described in Curtiss andEdgington, J. Biol. Chem., 257:15213-15221 (1982). Briefly, 50 μl of PBScontaining 5 μg/ml synthetic peptides were admixed into the wells ofmicrotiter plates. The plates were maintained overnight (about 16 hours)at 4° C. to permit the peptides to adhere to well walls. After washingthe wells four times with SPRIA buffer (2.68 mM KCL, 1.47 mM KH₂ PO₄,137 mM NaCl, 8.03 mM Na₂ HPO₄, 0.05% Tween-20, 0.1 KIU/ml Traysol, 0.1%BSA, 0.015% NaN₃), 200 μl of SPRIA buffer containing 3% normal goatserum (NGS) and 3% bovine serum albumin (BSA) were admixed to each wellto block excess protein binding sites. The plates were maintained for 30minutes at 20° C., the wells emptied by shaking, and blotted dry to forma solid-support, i.e., a solid matrix to which an apo E polypeptide wasoperatively affixed.

To each well was then admixed 50 μl of a serum supernatant containinganti-peptide antibodies for testing by SPRIA form a solid-liquid phaseimmunoreaction admixture. The admixture was maintained for 2 hours at37° C. to permit formation of solid-phase immunoreaction products. Afterwashing the wells as previously described, 50 μl of a second, revealantibody, ¹²⁵ I-labeled goat anti-mouse IgG, at 0.25 μg protein per mlwere admixed to each well to form a labeling reaction admixture. Thatadmixture was maintained for 1 hour at 37° C. to permit formation of ¹²⁵I-labeled solid-phase immunoreaction products. After washing the wellsas previously described, the amount of ¹²⁵ I-labeled product bound toeach well was determined by measuring gamma radiation from the labeledproduct. Specific anti-peptide antibody titers in collected serumsamples from immunized rabbits were thus determined and compared totiters measured using pre-immunized normal rabbit serum samples whichare a measure of non-specific background. Serum samples are consideredto contain anti-peptide polyclonal antibodies if the radioactive signalwas 5 times greater than seen with normal rabbit serum.

Anti-apo E polypeptide antibodies were obtained by the above procedurewhen peptide-LDL immunogen was used that contains the apo E peptidep(141-155)₂.

Additional anti-apo E polypeptide antibodies are prepared usingpeptide-LDL immunogens based on the apo E peptides p(141-155)₃ andself-conjugates of p(141-155)₂ and p(141-155)₃.

The above-described assay is utilized to determine whether thispolyclonal antisera binds apo E/VLDL. Apo E/VLDL is prepared asdescribed in Example 14a above. Instead of synthetic peptides, 50 ul ofPBS containing 5 μg/ml apo E/VLDL is admixed into the wells of themicrotiter plates. SPRIA assays performed in this manner indicate thatthe antibodies bind apo E/VLDL.

15. Monoclonal Antibody Preparation

a. Generation of Hybridomas

Balb/c ByJ mice (Scripps Clinic and Research Foundation Vivarium, LaJolla, Calif.) are immunized intraperitoneally (i.p.) with 50 μg oflipoprotein (polypeptide-carrier complex is used in the case of apo E incomplete Freund's adjuvant (CFA) followed by a second immunization inlipoprotein buffer on day 28, 3 days prior to fusion.

The animals immunized with apo E multimeric polypeptide analog,(p141-155)₂, conjugated to LDL as described in Example 14a, aresacrificed and the spleen of each mouse is harvested. A spleen cellsuspension is then prepared. Spleen cells are then extracted from thespleen cell suspension by centrifugation for about 10 minutes at 1000r.p.m., at 23 degrees C. Following removal of supernatant, the cellpellet is resuspended in 5 ml cold NH₄ Cl lysing buffer, and isincubated for about 10 minutes.

To the lysed cell suspension are admixed 10 ml Dulbecco's Modified EagleMedium (DMEM) (GIBCO) and HEPES[4-(2-hydroxyethyl)-1-piperidineethanesulfonic acid] buffer, and thatadmixture is centrifuged for about 10 minutes at 1000 r.p.m. at 23degrees C.

The supernatant is decanted, the pellet is resuspended in 15 ml of DMEMand HEPES, and is centrifuged for about 10 minutes at 1000 r.p.m. at 23degrees C. The above procedure is repeated.

The pellet is then resuspended in 5 ml DMEM and HEPES. An aliquot of thespleen cell suspension is then removed for counting. Fusions areaccomplished in the following manner using the non-secreting mousemyeloma cell line P3X63Ag8.653.1, a subclone of line P3x63Ag 8.653 (ATCC1580). Using a myeloma to spleen cell ratio of about 1 to 10 or about 1to 5, a sufficient quantity of myeloma cells are centrifuged into apellet, washed twice in 15 ml DMEM and HEPES, and centrifuged for 10minutes at 1000 r.p.m. at 23 degrees C.

Spleen cells and myeloma cells are combined in round bottom 15 ml tubes.The cell mixture is centrifuged for 10 minutes at 1000 r.p.m. at 23degrees C., and the supernatant is removed by aspiration. Thereafter,200 ul of 50 percent (weight per volume) aqueous polyethylene glycol4000 molecular weight (PEG; ATCC Baltimore, Md.) at about 37 degrees C.are admixed using a 1 ml pipette with vigorous stirring to disrupt thepellet, and the cells are gently mixed for between 15 and 30 seconds.The cell mixture is centrifuged 4 minutes at 700 r.p.m.

At about 8 minutes from the time of adding the PEG, 5 ml of DMEM plusHEPES buffer are admixed slowly to the pellet, without disturbing thecells. After 1 minute, the resulting admixture is broken up with a 1 mlpipette, and is incubated for an additional 4 minutes. This mixture iscentrifuged for 7 minutes at 1000 r.p.m. The supernatant is decanted, 5ml of HT (hypoxanthine/thymidine) medium are slowly admixed to thepellet, and the admixture is maintained undisturbed for 5 minutes. Thepellet is then broken into large chunks, and the final cell suspensionis placed into T75 flasks (2.5 ml per flask) into which 7.5 ml HT mediumhad been placed previously. The resulting cell suspension is incubatedat 37 degrees C to grow the fused cells. After 245 hours 10 ml of HTmedium are admixed to the flasks, followed 6 hours later by admixture of0.3 ml of 0.04 mM aminopterin. 48 hours after fusion, 10 ml of HAT(hypoxanthine/ aminopterin/thymidine) medium are admixed to the flasks.

Three days after fusion, viable cells are plated out in 96-well tissueculture plates at about 2×10⁴ viable cells per well (768 total wells) inHAT buffer medium as described in Kennett et al., Curr. Top. Microbiol.Immunol., 81:77 (1978). The cells are fed seven days after fusion withHAT medium and at approximately 4-5 day intervals thereafter as neededwith HT medium. Growth was followed microscopically, and culturesupernatants were collected about two weeks later and assayed for thepresence of antibody molecules that immunoreact with the immunizingpolypeptide by solid phase radioimmunoassay (SPRIA) as described inExample 14b.

Briefly, 50 ul of PBS containing 5 μg/ml of the immunizing apo Epolypeptide of the immunizing apo E polypeptide, (p141-155)₂, areadmixed into the wells of microtiter plates. The plates are maintainedfor 3 hours at room temperature to permit the polypeptide to adhere towell walls. After washing the wells four times with SPRIA buffer (2.68mM KCl, 1.47 mM KH₂ PO₄, 137 mM NaCl, 8.03 mM Na₂ HPO₄, 0.05% Tween-20,0.1 KIU/ml Traysol, 0.1% BSA, 0.015% NaN₃), 200 ul of SPRIA buffercontaining 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA)are admixed to each well to block excess protein binding sites. Theplates are maintained for 30 minutes at 20 degrees C., the wells emptiedby shaking, and blotted dry to form a solid-support, i.e., a solidmatrix to which apo E polypeptide was operatively affixed.

To each well containing an immunizing polypeptide was then admixed 50 ulof hybridoma tissue culture supernatant produced by the correspondingimmunizing polypeptide to form a solid-liquid phase immunoreactionadmixture. The admixture is maintained for 2 hours at 37 degrees C. topermit formation of solid-phase immunoreaction products. After washingthe wells as previously described, 50 ul of ¹²⁵ I-labeled goatanti-mouse IgG at 0.25 μg protein per ml are admixed to each well toform a labeling reaction admixture. That admixture is maintained for 1hour at 37 degrees C. to permit formation of ¹²⁵ I-labeled solid-phaseimmunoreaction products. After washing the wells as previouslydescribed, the amount of ¹²⁵ I-labeled product bound to each well wasdetermined by measuring gamma radiation from the labeled product.Hybridoma culture supernatants are considered to contain monoclonalanti-apo E polypeptide antibodies if the immunoreaction product formedproduced a radioactive signal five times greater than a signal measuredusing a control hybridoma culture supernatant.

The anti-B-100 monoclonal antibody MB47 was prepared essentially asdescribed above, except that LDL was used as the immunogen, and theantibody was selected by screening for immunoreaction with isolated apoB-100.

b. Isolation of Immunoglobulin

Ascites fluids containing monoclonal antibody molecules useful hereinwere prepared using 10-week-old Balb/c mice. The mice were first primedwith 0.3 ml of mineral oil and injected intraperintoneally with 3-50×10⁵MB47 hybridoma cells prepared as described in Example 15a. The averagetime for development of ascites was 12 days. The resulting ascites fluidwas collected and clarified by centrifugation at 15,000×g for 1 hour at4 degrees C. to form clarified ascites fluids, which can be pooled andstored if desired, at -20 degrees C.

Isolated MB47 monoclonal antibody molecules were prepared bychromatography of the clarified ascites fluids on a protein A-Sepharose4B column (Pharmacia Fine Chemicals, Piscataway, N.J.). Antibody waseluted from the column with 0.1 molar (M) acetic acid to form theisolated monoclonal antibody MB47.

Isolated monoclonal antibody molecules were also prepared by fastprotein liquid chromatography (FPLC) of a clarified ascites fluid on aPharmacia Mono Q HR 5/5 anion exchange column in a Pharmacia FPLC Systemusing a 0-0.5M NaCl gradient in 10 mM Tris, pH 8.0, and following thedirections supplied with the column. The resulting isolated monoclonalantibody molecules were concentrated using an Amicon stirredultrafiltration cell (Danvers, Mass.; PM 30 membrane) to a concentrationof 1 mg/ml, dialyzed into PBS (phosphate-buffered saline, pH 7.2) andstored at -70 degrees C. to form purified monoclonal antibody.

Monoclonal anti-apo E polypeptide antibody molecules can be purified asabove using the hybridoma that can be prepared by the methods of Example15a to form purified anti-apo E polypeptide monoclonal antibody.

16. Solid-Phase Polypeptide ELISA

Apo E polypeptides prepared in Example 1 immunoreact with an anti-apo Epolypeptide antibody in a direct binding ELISA. In the assay, 50 μg/mlof a polypeptide-containing solution of (p141-155)₂ is dissolved in PBSto form a peptide coating solution, of which 150 μl is admixed into thewells of a microtiter plate. The wells are then maintained for about 16to 20 h at 4° C. to permit the peptide to adsorb onto (coat) the wallsof the wells. After removing the peptide coating solution by shaking,the wells are washed once with 350 μl of rinsing buffer (PBS containing1 g/l BSA, 0.5 ml/l Tween 20, and 2 μl/l aprotinin). Excess proteinbinding sites are blocked by admixing 200 μl of blocking buffer (PBScontaining 3% BSA) into each well, maintaining the wells for 1 hour at37° C., removing the blocking buffer by shaking, and then washing thewells 3 times as previously described. The plate is then dried for 1hour at 37° C. followed by addition of 100 μl of PBS containing 0.5μg/ml horseradish peroxidase-conjugated anti-(p141-155)₂ antibody,prepared according to the method of Nakane, et al., J. Histochem.Cytochem., 22:1084 (1974), to form a solid-liquid phase immunoreactionadmixture. The resulting solid-liquid phase immunoreaction admixture ismaintained at 20° C. for 1 hour to permit formation of a solid-phasepolypeptide-containing immunoreaction product. The wells are then washed3 times with rinsing buffer to remove unbound antibody.

Assays are performed in identical manner using anti-p(141-155)₃polyclonal antibodies, which are elicited as previously described andconjugated to horseradish peroxide.

The amount of immunoreaction product present in the solid phase is thendetermined by admixing two hundred microliters of OPD substrate (3% H₂O₂ and 0.67 mg/ml o-phenylene diamine) into each well to form adeveloping-reaction admixture. The admixture is maintained for 30minutes at about 20° C. Subsequently, 50 μl of 4 N H₂ SO₄ are admixedinto each well to stop the developing-reaction, and the resultingsolution is assayed for absorbance at 450 nanometers using a microtiterplate reader (Dynatech) to detect the amount of formed immunoreactionproduct.

In order to determine specificity of the antibody molecules using thesame assay, a monomer peptide, p141-155, is prepared as described inExample 1 and tested as above. Additionally, control peptides p93-112and p172-182 are prepared and tested. The results in all cases indicatethat these antibodies do not bind either the monomer or the controlpeptides.

A competition ELISA is useful to detect apo E polypeptide or apo E in afluid sample such as serum. Microtiter plates are coated with the dimerp(141-155)₂ as described hereinbefore. After the drying step of theassay described hereinbefore, 50 μl of a fluid sample (i.e., an apo Epolypeptide-containing or an apo E-containing fluid sample) or standard(i.e., an apo E polypeptide as a standard) to be assayed are admixedinto the polypeptide-coated well simultaneously with 50 μl ofHRPO-conjugated antipeptide antibody to form an immunoreactionadmixture. In the assay described herein, two competitors (polypeptidestandard or the fluid sample) are tested in separate immunoreactionadmixtures for their ability to compete for binding of the anti-apo Eantibody to the coated polypeptide analog over a range of antibodydilutions. The polypeptide standard in solution is added at a startingconcentration of 1 mg/ml and serially diluted to a final concentrationof 0.0156 mg/ml. Serum or plasma fluid samples are added at a startingdilution of 1:10 and diluted serially to a final dilution of 1:320. Theplate is then incubated for 30 minutes at room temperature, washed andthe assay developed as described hereinbefore to determine the amount ofimmunoreaction product formed, and thereby the amount of competitorpresent in the added fluid sample.

The competition ELISA is particularly preferred to measure apoE-containing lipoprotein particles in bodily fluid, such as total apo E,and can also be used to monitor the fate of therapeutically administeredapo E polypeptide present in the serum of a patient after administrationof therapeutic apo E polypeptide compositions.

a. Apo E Sandwich Assay

Described herein is a capture or sandwich assay. Polystyrene microtiterplates (Nunc-Immuno Plate I) are coated with 150 ul of sodiumbicarbonate buffer, pH 9.0, containing 1 μg/ml of purifiedanti-p(141-155)₃ polyclonal antibody for 16 hours at 4° C. to prepare asolid-phase capture antibody. The plates are washed 3 times with PBScontaining 0.1% BSA, 0.05% Tween, and then blocked with 3% BSA exactlyas described above. The apo E/VLDL standard (prepared as in Example 14)is diluted 1:200 in PBS containing 0.5% (Diluting buffer=LPDP/PBS) toconcentrations ranging from 0.125 to 4.0 μg/ml. The same controls asdescribed above for the competition ELISA are used in this assay. Plasmaor serum samples and controls are diluted 1000-fold in dilution buffer.Fifty ul of standards and unknown samples are added to the wells intriplicate.

The plates are incubated exactly 30 minutes at 25° C., and the liquidphase is removed from the wells. Fifty ul of PBS, containing a fixedconcentration of HRPO-linked anti-B100 monoclonal reveal antibody(MB47), described before, is immediately pipetted into wells containingaliquot 1 of the plasma samples. The plates are incubated exactly 30minutes at 25° C., washed, and 100 ul of OPD substrate solution areadded for an additional 30 minute incubation at 25° C. Color developmentis stopped by addition of 50 ul of 4N H₂ SO₄ and plates are read in amicroplate reader as described before.

17. Effect of Multimers of 141-155 Sequence on Lymphocyte Proliferation

The effects of mono-, di-, tri- and tetra-mers of the p(141-155) peptideon lymphocyte proliferation were studied following the assay describedin Example 2. As shown in FIG. 4, monomeric p(141-155) had no effect onlymphocyte proliferation over the range of peptide concentrationsexamined.

On the other hand, the di-, tri- and tetramers of p(141-155) each showedan ability to inhibit lymphocyte proliferation at higher peptideconcentrations. Calculation of an ED₅₀ or dose of peptide required toinhibit lymphocyte proliferation 50% for the dimer, trimer and tetramerwas 20, 7, and 1.2 μM, respectively. Therefore, the inhibiting effect ofthe multimeric polypeptides on lymphocyte proliferation increased withthe multiplicity (repeat number) of the p(144-155) sequence in thepolypeptide. The formulas of the apo E peptides studied were: monomer,p(141-155); dimer, p(141-155)₂ ; trimer, p(141-155)₃ ; tetramet,p(141-155)₄.

18. Effect of Amino Acid Substitutions on Lymphocyte Proliferation

The effect on lymphocyte proliferation of single amino acidsubstitutions in both repeats of the apo E 141-155 sequence in thetandem peptides was studied.

The proliferation assays were performed as described in Example 2 andthe substituted tandem polypeptides were identical to those described inExample 10, i.e., Lys 143 substituted by Ala, Arg 150 substituted byAla, and Leu 144 substituted by Pro. The results are presented in FIG.5, along with the results for unsubstituted tandem p(141-155)₂ peptide.

Each of the active peptides studied showed at least some indication ofbimodal activity, i.e., proliferation enhancement at low concentrationsand inhibition at higher concentrations, as is found in native apo E.The Leu(144) to Pro substitution substantially reduced the biologicalactivity of the tandem peptide, whereas the other two substitutionsappeared to have little effect on the biologic activity.

19. Identification of Additional Apo E Polypeptides

Lipoproteins containing apoprotein (apo) E inhibit mitogen- or antigen-stimulated lymphocyte proliferation both in vitro and in vivo. Curtisset al, J. Immunol., 118:1966-1970 , (1977); and Curtiss et al, J.Immunol., 118:648-652, (1977). Apo E has been shown to be a keycomponent, since lipid-free apo E has been shown to have comparableactivity. Pepe et al, J. Immunol., 136:3716-3723, (1986). In the presentdisclosure, the active site of apo E that is responsible for its abilityto inhibit lymphocyte proliferation has been identified. A syntheticdimeric repeat of amino acid residues 141-155 of human apo E was able tosuppress PHA-stimulated lymphocyte proliferation in a manner that wasanalogous to native apo E. The apparent requirement for duplication ofthe 141-155 sequence was demonstrated by observing that the monomericversion of this sequence was not active.

Why only a duplicated 141-155 domain in the form of multimers or aself-conjugate, but not monomeric conjugates to a carrier, is active isunknown. Therefore, to explore in greater depth the structuralrequirements for function and biologic activity, a number of modifiedpeptides were synthesized and purified. Studies specifically addressingthe structural requirements for the biologic activity of the syntheticpeptide mimic were then conducted as described below. Included is anassessment of the effects of modification of specific amino acidresidues and the effects of increasing the number of duplications.Finally, the effects of specific amino acid deletions were examined inan attempt to identify a minimum functional sequence for an LDL-receptorbinding moiety. With molecular modeling and energy minimizationanalyses, the importance of various structural features of the peptidesfor biologic activity was assessed as well.

In these studies, human peripheral blood mononuclear (PBM) cells wereisolated and assayed for phytohemagglutinin (PHA)-stimulatedproliferation as described by Pepe et al (J. Immunol., 136:3716-3723,1986) except that the cells were resuspended and cultured in aserum-free and protein-free CFBI medium as described by Shive et al,Proc. Natl. Acad. Sci. USA, 83:9-13, (1986). The PBM cells (200,000 in0.2 ml) were incubated at 37° C. with 0.05 ml of peptide and PHA. After48 h the cultures were pulsed for 18 h with 0.5 mCi of [³H]-methylthymidine and harvested onto glass fiber filters. Radioactivityof the filters was counted in a Beckman liquid scintillation counter.Results were expressed as mean cpm ±S.D. or as percent inhibition thatwas derived from 100×(1 -cpm of test culture/cpm of control culture).All data shown are representative of a single experiment performed inquadruplicate and all experiments were repeated at least 3 times toverify that the results reported were reproducible. The averagebiological activity of each polypeptide for inhibiting lymphocyte (PMN)proliferation is shown in Table 6, and is expressed as an averagespecific activity, which is the peptide concentration that causes a 50%reduction in activity.

PBM cell viability following exposure to the peptides was assessed bymeasuring lactate dehydrogenase (LDH) release using a Sigma diagnosticLDH assay kit as described in Example 3. Following culture the cellsupernatants were diluted 10-fold in 0.1 M sodium phosphate buffer, pH7.4 and 6 mM NADH. Absorbance was measured after the addition of 6 mMsodium pyruvate and measured kinetically at 340 nm. Relativeconcentration of LDH in the culture supernatants was compared to totalLDH release obtained by freeze and thaw lysis of the cells in water.None of the peptides shown in Table 6 exhibited significant cytolysis.

Studies of the effect of charge on the capacity of native apo E to bindto the LDL receptor have demonstrated the importance of positivelycharged basic amino acids. Mahley et al, J. Biol. Chem., 252:7297-7287,(1977); and Wilson et al, Science, 252:1817-1822, (1991). There are 12lysine and arginine residues in the dimer peptide (Table 6). To test theimportance of these basic amino acids for biologic activity, the lysineresidues of the dimer were modified by acetylation and the arginineresidues by cyclohexanedione modification. As expected, each of thesemodifications abolished the biologic activity of the tandem peptide,indicating the importance of the positively charged basic amino acidresidues.

                                      TABLE 6                                     __________________________________________________________________________                                # Amino  Average Specific                         Peptide                                                                              Peptide #                                                                           Sequence       Acids                                                                              Purity                                                                            Activity (μM)                                                                       n.sup.1                                                                         Total Energy                  __________________________________________________________________________    Monomer                                                                              (15)  LRKLRKRLLRDADDL                                                                              15       None     0 -344.08 ± 0.01             Dimer  53    (LRKLRKRLLRDADDL).sub.2                                                                      30   93  4.8 ± 1.1                                                                           8 -689.57 ± 0.04             143 lys--ala                                                                         36    (LRALRKRLLRDADDL).sub.2                                                                      30   99  5.6 ± 1.7                                                                           3 -708.357 ± 0.021           150 arg--ala                                                                         38    (LRKLRKRLLADADDL).sub.2                                                                      30   98  7.5 ± 3.0                                                                           3 -503.34 ± 0.05             144 leu--pro                                                                         37    (LRKPRKRLLRDADDL).sub.2                                                                      30   98  None     3 -624.390 ± 0.018           129-162                                                                               0    STEELRVRLASHLRKLRK-                                                                          35   98  None     3 -457.99 ± 0.05                          RLLRDADDLQKRLAVYQ                                                Tetramer                                                                             35    Y(LRKLRKRLLRDADDL).sub.4                                                                     60   99  2.8 ± 1.3                                                                           4 -1418.30 ± 0.01            (141-150).sub.2                                                                      46    (LRKLRKRLLR).sub.2                                                                           20   99  4.9 ± 1.2                                                                           3 -587.32 ± 0.03             (145-155).sub.2                                                                      43    (RKRLLRDADDL).sub.2                                                                          22   92  None     3 -581.14 ± 0.05             (144-150).sub.2                                                                      30    (LRKRLLR).sub.2                                                                              14   94  None     3 -455.71 ± 0.10             (LLRK).sub.4                                                                         47    (LLRK).sub.4   16   99  1.6 ± 0.5                                                                           6 -232.66 ± 0.09             (LLRK).sub.8                                                                         45    (LLRK).sub.8   32   95  1.1 ± 0.4                                                                           5 -483.55                       __________________________________________________________________________                                                    ± 0.09                      .sup.1 number of times assayed                                           

The effect of specific single amino acid substitutions of lysine andarginine residues was also examined by preparing synthetic dimericrepeats of the 141-155 peptide with substitutions of an alanine forlysine 143 or an alanine for arginine 150 (Table 6). All single aminoacid substitutions were made at both positions in the duplicated 141-155sequence. The effect of these substitutions on biologic activity wastested in the above assay for inhibition of lymphocyte proliferation.The results were that over a peptide concentration of 0.2 to 30micromolar (uM), p(141-155)₂, 143 lys-ala substitution peptide and 150argala substitution peptide inhibited uptake of tritiated thymidineabove about 4 uM. Slight differences in the specific biologic activitywere observed. However, neither of these single amino acid substitutionshad a significant effect on the activity of the dimer.

In the contrast to the minimal effects seen with the single lysine orarginine substitutions, another single amino acid substitution, had asignificant effect on its biologic activity. Substitution of a prolinefor leucine 144 essentially eliminated the biologic activity. Thissubstitution would be expected to have a profound effect on the α-helixforming properties of this peptide suggesting that the helix structureof this peptide may be important for activity.

The importance of maintenance of α-helical structure for biologicactivity also was examined by preparing another peptide. This peptide,which represented a single copy of amino acid residues 129-162 of humanapo E (Table 6), has been determined by x-ray crystallography torepresent the entire fourth α-helix of a four helical bundle withinintact apo E. Wilson et al, Science, 252:1817-1822, (1991). Computermodeling of this extended monomeric peptide suggests that the α-helicalnature of this region of the protein is quite stable. However, whenmeasured for inhibition of lymphocyte proliferation as above, p(129-162)exhibited no biologic activity. The results suggest that the α-helixstructure of this region is essential but not sufficient for biologicactivity.

Many of the biologic functions of native apo E are best accomplished bylipoprotein particles that contain multiple copies of this apoprotein.Mahley et al, Biochim. Biophys. Acta., 737:197-222, (1983); and Kowal etal, Proc. Natl. Acad. Sci. U.S.A. 86:5810-5814, (1989). This also hasbeen shown to be true for the immunosuppressive activity of apo EHDL_(C). Hui et al, J. Biol. Chem., 255:11775-11781, (1980). None of themonomer peptides has biologic activity, whereas the dimer is inhibitoryat a concentration range of between 2 and 5 μM. If the multimeric natureof the peptide is lay to its activity, the possibility exists that wecould enhance activity even further by making larger multimers of the141-155 sequence. To test the concept that peptides with more than 2copies of this sequence would have greater biologic activity, a tetramerwas synthesized and purified (Table 6). As shown in Table 6, thetetrameric representation of the 141-155 sequence had twice theinhibitory activity of the dimer on a molar basis. Therefore, increasingthe number of repeats of the 141-155 sequence in a peptide enhanced itsinhibitory activity. A dose-response curve for inhibition of lymphocyteproliferation by the dimer, trimer and tetramer is shown in FIG. 4.

More reiterations of the 141-155 sequence exhibited greater biologicactivity. However, 60 residues rapidly approached the size limit for thesynthesis of peptides of the high purity and integrity needed for anaccurate quantitative assessment of function. Therefore, to identify aminimum dimeric sequence for biologic activity, three additionalpeptides were synthesized and their biologic activities compared withthe parent dimer. The first peptide was a dimeric repeat of amino acidresidues 141-150. This peptide did not contain the last five carboxyterminal residues of the 141-155 dimer repeat, DADDL (Table 6).Interestingly, peptide p(141-150)₂ had an activity that was comparableto that of the 141-155 dimer, inhibiting lymphocyte proliferation atconcentrations above about 3 uM. In fact its average specific activityof 4.9±1.2 μM was very similar to that calculated for the 141-155 dimer,4.8±1.1 μM (Table 6). This indicates that the aspartic acid-richsequence carboxy-terminal portion of the dimer is not important foractivity.

Further support for the importance of the first ten residues in the141-155 sequence came from assay of a dimeric repeat of amino acidresidues 145-155. This peptide did not contain the amino terminal LRKLsequence while retaining the carboxy terminal aspartic acid-richsequence, DADDL. Removal of the LRKL residues in the form of peptidep(141-155)₂ resulted in the loss of biologic activity and confirmed therelative unimportance of the DADDL residues for biologic activity.Confirmation of the absolute requirement for the amino terminal LRKresidues was obtained by preparation of a peptide that represented arepeat of amino acid residues 144-150 (Table 6). Peptide p(144-150)₂ didnot contain the amino terminal LRK residues as well as thecarboxy-terminal DADDL residues. As for the previous peptide, thisdimeric repeat had no biologic activity at concentrations of > 210 μM.Therefore, the residues that were key to the biologic activity includethe leucine-, arginine- and lysine-rich sequence, LRKLRKRLLR.

Inhibition of lymphocyte proliferation measured as described above usingthe polypeptides in Table 6, namely p(141-155)₂, p(144-150)₂,p(145-155)₂ and p(141-150)₂, yields a dose-response of inhibition forthese polypeptides, and is shown in FIG. 6.

To further explore the importance of this leucine-, arginine- andlysine-rich motif, two additional peptides were synthesized and testedfor biologic activity. Peptides containing four and eight repeats of theLLRK sequence were prepared (Table 6) and both of these peptidesinhibited PHA- stimulated thymidine uptake. The average specificactivity of the (LLRK)₄ repeat from 6 separate experiments was 1.6±0.5μM, whereas the (LLRK)₈ repeat had an average specific activity of1.1±0.4 μM. Inhibition of lymphocyte proliferation measured as describedabove using the polypeptides p(141-155)₂, p(LLRK)₄, p(LLRK)₈ andp(141-150)₂, yields a dose-response of inhibition for thesepolypeptides, and is shown in FIG. 7. Thus, the biologic activity of thereiterated LLRK sequence adds further support to the importance of theregion defined by amino acid residues 141-145 of native apo E forbiologic activity. Furthermore, these observations support the apparentrequirement for reiteration of this motif.

To verify that the activity seen with each of the inhibitory syntheticpeptides was not a result of direct cellular toxicity, LDH assays asdescribed in Example 3 were performed on all culture supernatants fromthe lymphocyte cultures. None of the peptides exhibited significant(less than 5%) release of LDH activity, indicating that the peptideswere not cytotoxic.

Although the present invention has been described in terms of certainpreferred embodiments, various modifications, changes, omissions andsubstitutions may be made without departing from the spirit thereof.

What is claimed is:
 1. A synthetic polypeptide consisting of two to tensegments each having an amino acid sequence of theformula:Leu-Arg-Xaa-Leu-Arg-Lys-Arg-Leu-Leu-Xbb,where Xaa is Lys or Ala,and Xbb is Arg or Ala, said polypeptide having the ability to inhibitlymphocyte proliferation.
 2. The polypeptide of claim 1 wherein one ofsaid segments has an amino acid sequence corresponding to a formulaselected from the group consisting of:(1)Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg, (2)Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Arg, (3)Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Ala, and (4)Leu-Arg-Ala-Leu-Arg-Lys-Arg-Leu-Leu-Ala.
 3. The polypeptide of claim 1wherein said polypeptide has an amino acid sequence selected from thegroup consisting of: ##STR1##
 4. A synthetic polypeptide consisting ofan amino acid sequence corresponding to a formula selected from thegroup consisting of: ##STR2## said polypeptide having the ability toinhibit lymphocyte proliferation.